Methods and compositions for prevention and treatment of hearing loss

Abstract

The present disclosure provides methods of preventing and / or treating hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. The present disclosure also provides methods of preventing and / or treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

Description

Docket No.: 336122000640METHODS AND COMPOSITIONS FOR PREVENTION AND TREATMENT OF HEARING LOSSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 725,951, filed November 27, 2024, entitled “METHODS AND COMPOSITIONS FOR PREVENTION AND TREATMENT OF HEARING LOSS,” which is herein incorporated by reference in its entirety for all purposes.FIELD

[0002] The presently disclosed relates generally to methods of preventing and / or treating hearing loss and / or a vestibular disorder in an individual by increasing the expression and / or activity of a Pou4f3 protein.REFERENCE TO AN ELECTIONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (336122000640SeqList.xml; Size: 10,543 bytes; and Date of Creation: November 25, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Hearing loss is one of the most prevalent sensory deficits affecting more than 450 million people worldwide. At present, there is no FDA-approved drug with demonstrated efficacy to improve this life-altering situation. Further, individuals with hearing loss are more likely to have vestibular disorders than those who do not have hearing loss.

[0005] Hearing loss disproportionately affects human patients. Hearing loss, due to the loss of ability to detect sound, is generally permanent. There are different degrees of hearing loss, including mild, moderate, severe and profound, measured by ABR (Auditory Brainstem Response) threshold shift. Hearing impairment can also manifest as deficiencies in word recognition, especially in noisy environments. The mechanisms underlying different types of hearing loss are different. The damage to and death of hair cells, the inner ear sensory cells that detect sounds, is one of the major causes of permanent hearing loss. The loss of synapses, termed synaptopathy, between inner hair cells and spiral ganglion neurons (SGN) is likely the underlying cause of word recognition deficiency.1MF-364562974Docket No.: 336122000640

[0006] Hearing functions through the detection of mechanical vibrations by inner ear sensory hair cells, which release neurotransmitters that are absorbed by the auditory spiral ganglion neurons, which generate electric signals and pass them into the brain. The cells (including hair cells and ganglion neurons) and their structures (stereocilia of hair cells, synapses between hair cells and SGN, and neurites of SGN) along the path of sound detection and transmission can be damaged by noise, ototoxic drugs, viral infection, gene mutations, and aging, which ultimately lead to hearing impairment. Noise-induced hearing loss (NIHL) and age-related hearing loss (ARHL) are the two main categories of hearing loss.

[0007] Therapeutics that can repair damaged cochlear hair cells and synapses to restore functions are promising avenues to reconstitute the auditory pathways and treat hearing loss. In addition, therapeutics that can prevent or slow down the progression of hearing loss could be effective interventions to benefit numerous patients with sudden hearing loss or progressive age-related hearing loss.

[0008] While distinct mechanisms exist for different forms of hearing loss such as NIHL, ARHL, and word recognition deficiency, therapeutics that target one mechanism may have treatment effect on more than one form of hearing loss. Repair of damaged hair cells by an intervention to restore hearing may also improve synaptic function, which can be applied to word recognition deficiency.

[0009] Accordingly, there is an unmet need for therapies that can treat and prevent hearing loss and vestibular disorders. Provided herein are methods that meet such needs.BRIEF SUMMARY

[0010] At present, there is no FDA-approved drug with demonstrated efficacy to treat noise- induced hearing loss (NIHL) and age-related hearing loss (ARHL). Demonstrated herein, for the first time, the overexpression of the key transcription factor, Pou4f3, in the inner ear hair cell is efficacious to attenuate noise induced hearing loss (NIHL), repair synaptopathy, and ameliorate progressive hearing decline in age-related hearing loss (ARHL). In particular, it was shown that overexpression of Pou4f3 can aid in the repair of hair cells, hair cell stereocilia, and hair cell synaptic ribbons. This contrasts with prior studies, demonstrating that co-expression of transcription factors, for example Atohl, Gfil, Pou4f3, and Sixl, can induce hair cell-like regeneration (e.g., Zhang, L. et al. AAV-mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration. Adv Sci (Weinh ) 11, e2304551 (2024)). However, inducing regeneration of immature hair cells has not been shown to restore hearing and / or treat2MF-364562974Docket No.: 336122000640 any type of hearing loss. Accordingly, the present disclosure provides methods of preventing and / or treating hearing loss in an individual by increasing the expression and / or activity of a Pou4f3 protein in the individual, and overexpression of Pou4f3 alone in the absence of other transcription factors (Atohl, Gfil, and Six) is sufficient to repair damaged hair cells and improve hearing. The present disclosure also provides methods of preventing and / or treating a vestibular disorder in an individual by increasing the expression and / or activity of a Pou4f3 protein in the individual.

[0011] In some aspects, provided herein is a method of preventing and / or treating hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0012] In some aspects, provided herein is a method of preventing and / or treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0013] In some embodiments, the agent is a Pou4f3 protein.

[0014] In some embodiments, the agent is a nucleic acid encoding a Pou4f3 protein. In some embodiments, the nucleic acid encoding the Pou4f3 protein is an RNA molecule. In some embodiments, the nucleic acid encoding the Pou4f3 protein is a DNA molecule.

[0015] In some embodiments, the agent activates a Pou4f3 gene. In some embodiments, the agent is a small activating RNA. In some embodiments, the agent is a small molecule.

[0016] In some aspects, provided herein is a method of preventing and / or treating hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein.

[0017] In some aspects, provided herein is a method of preventing and / or treating a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein.

[0018] In some embodiments, the composition comprises a viral vector comprising the nucleic acid sequence encoding the Pou4f3 protein.

[0019] In some embodiments, the composition is administered to the ear of the individual. In some embodiments, the composition is administered to a cochlear cell in the ear of the individual. In some embodiments, the cochlear cell is selected from the group consisting of a stria vascularis cell, a hair cell, a supporting cell, and a spiral ganglion neuron. In some embodiments, the cochlear cell is a hair cell. In some embodiments, the hair cell is an inner hair cell. In some embodiments, the hair cell is an outer hair cell.3MF-364562974Docket No.: 336122000640

[0020] In some embodiments, the agent is a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein.

[0021] In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a promoter sequence. In some embodiments, the promoter sequence is selected from the group consisting of a constitutive promoter sequence, an inducible promoter sequence, and a tissue- specific promoter sequence. In some embodiments, the promoter sequence is a constitutive promoter sequence. In some embodiments, the constitutive promoter sequence is selected from the group consisting of a human cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, a simian virus 40 (SV40) promoter sequence, and a mammalian elongation factor la (EFla) promoter sequence. In some embodiments, the constitutive promoter sequence is a CMV promoter sequence. In some embodiments, the promoter sequence is a tissue-specific promoter sequence. In some embodiments, the tissue-specific promoter sequence is selected from the group consisting of a stria vascularis cell-specific promoter sequence, a hair cell-specific promoter sequence, a supporting cell- specific promoter sequence, and a spiral ganglion neuron- specific promoter sequence.

[0022] In some embodiments, the viral vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vesicular stomatitis virus (VSV) vector, a herpes simplex virus (HSV) vector, a vaccinia virus vector, a pox virus vector, an influenza virus vector, a respiratory syncytial virus vector, a parainfluenza virus vector, a foamy virus vector, and a retrovirus vector. In some embodiments, the viral vector is an adeno-associated virus type 2 (AAV2) vector.

[0023] In some embodiments, the Pou4f3 protein encoded by the nucleic acid sequence comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Pou4f3 protein encoded by the nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 4.

[0024] In some embodiments, the composition is administered directly to the inner ear of the individual. In some embodiments, the composition is administered through the posterior semicircular canal of the ear of the individual. In some embodiments, the composition is administered through the round window membrane of the ear of the individual. In some embodiments, the composition is administered to both ears of the individual.

[0025] In some embodiments, the method further comprises determining the auditory brainstem response (ABR) threshold of the individual at a frequency before and after the4MF-364562974Docket No.: 336122000640 individual has received the composition. In some embodiments, the method results in a decrease in the ABR threshold of the individual at a frequency as compared to the ABR threshold of the individual at the same frequency prior to administration of the composition.

[0026] In some embodiments, the method further comprises determining the Wave I amplitude of the individual at a frequency before and after the individual has received the composition. In some embodiments, the method results in an increase in the Wave I amplitude of the individual at a frequency as compared to the Wave I amplitude of the individual at the same frequency prior to administration of the composition.

[0027] In some embodiments, the method further comprises determining the distortion product otoacoustic emission (DPOAE) threshold of the individual at a frequency before and after the individual has received the composition. In some embodiments, the method results in a decrease in the DPOAE threshold of the individual at a frequency as compared to the DPOAE threshold of the individual at the same frequency prior to administration of the composition.

[0028] In some embodiments, the method reduces and / or prevents the loss of outer hair cells and / or inner hair cells in the ear of the individual. In some embodiments, the method reduces and / or prevents the loss of synapses between inner hair cells and spiral ganglion neurons in the ear of the individual. In some embodiments, the method reduces and / or prevents the loss of stereocilia in the ear of the individual.

[0029] In some embodiments, the hearing loss is noise-induced hearing loss. In some embodiments, the hearing loss is age-related hearing loss. In some embodiments, the hearing loss is progressive non-syndromic hearing loss (NSHL). In some embodiments, the individual has a mutation in a POU4F3 gene. In some embodiments, the hearing loss is progressive hearing loss. In some embodiments, the individual has a mutation in a hair cell bundle gene. In some embodiments, the individual has a mutation in a CDH23 gene.

[0030] In some embodiments, the vestibular disorder is associated with hearing loss in the individual. In some embodiments, the vestibular disorder is associated with progressive hearing loss. In some embodiments, the vestibular disorder is associated with age-related hearing loss.

[0031] In some embodiments, the individual exhibits cochlear synaptopathy. In some embodiments, the individual exhibits tinnitus.

[0032] In some embodiments, the individual is human.

[0033] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the5MF-364562974Docket No.: 336122000640 art. These and other embodiments of the invention are further described by the detailed description that follows.DESCRIPTION OF THE FIGURES

[0034] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0035] FIG. 1 shows a schematic of a study design for assessing the treatment of 110 decibel (dB) sound pressure level (SPL) noise damage in CBA / CaJ mice with an AAV-Pou4f3 expression vector.

[0036] FIG. 2 shows a summary of the auditory brainstem response (ABR) Wave 1 amplitude data for four CBA / CaJ mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in treated and untreated contralateral ears 1 month after AAV-Pou4f3 treatment. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak’s multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0037] FIG. 3 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for four CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 1 month after AAV-Pou4f3 treatment for 1 lOdB permanent threshold shift (PTS) noise damage. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0038] FIGs. 4A-4B shows the average auditory brainstem response (ABR) (FIG. 4A) and distortion product otoacoustic emission (DPOAE) (FIG. 4B) thresholds for four CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 1 month after AAV-Pou4f3 treatment for 110 dB permanent threshold shift (PTS) noise damage. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01. Values and error bars reflect mean ± SEM.

[0039] FIG. 5 shows a summary of the auditory brainstem response (ABR) Wavel amplitude data at 100 dB for four CBA / CaJ mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with6MF-364562974Docket No.: 336122000640Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0040] FIG. 6 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for four CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment for 1 lOdB permanent threshold shift (PTS) noise damage. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001=. Values and error bars reflect mean ± SEM.

[0041] FIGs. 7A-7B shows the average auditory brainstem response (ABR) (FIG. 7A) and distortion product otoacoustic emission (DPOAE) (FIG. 7B) thresholds for four CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment for 110 dB permanent threshold shift (PTS) noise damage. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0042] FIG. 8 shows a schematic of a study design for assessing protection from permanent threshold shift (PTS) noise induced hearing loss in CBA / CaJ mice by an AAV-Pou4f3 therapy.

[0043] FIG. 9 shows a summary of the auditory brainstem response (ABR) Wave 1 amplitudes data for four CBA / CaJ mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in AAV-Pou4f3 pretreated and untreated contralateral ears 11.5 weeks after lOOdB sound pressure level (SPL) noise exposure. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test : *p < 0.05, **p < 0.01. Values and error bars reflect mean ± SEM.

[0044] FIG. 10 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for four CBA / CaJ mice at multiple frequencies (kHz) in AAV-Pou4f3 pretreated and untreated contralateral ears 11.5 weeks after HOdB permanent threshold shift (PTS) noise damage. N=4 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0045] FIGs. 11A-11B shows the average auditory brainstem response (ABR) (FIG. 11A) and distortion product otoacoustic emission (DPOAE) (FIG. 11B) thresholds for four CBA / CaJ mice at multiple frequencies (kHz) in AAV-Pou4f3 pretreated and untreated contralateral ears 11.5 weeks after HOdB permanent threshold shift (PTS) noise damage. N=47MF-364562974Docket No.: 336122000640 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0046] FIG. 12 shows a schematic of a study design for assessing the treatment of age-related hearing loss in C57Bl / 6j mice with an AAV-Pou4f3 therapy.

[0047] FIG. 13 shows a summary of the auditory brainstem response (ABR) Wave 1 amplitude data for five C57Bl / 6j mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in treated and untreated contralateral ears 2 months after AAV-Pou4f3 treatment. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0048] FIG. 14 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for five C57Bl / 6j mice at multiple frequencies (kHz) in treated and untreated contralateral ears 2 months after AAV-Pou4f3 treatment. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001. Values and error bars reflect mean ± SEM.

[0049] FIGs. 15A-15B shows the average auditory brainstem response (ABR) (FIG. 15A) and distortion product otoacoustic emission (DPOAE) (FIG. 15B) thresholds for five C57Bl / 6j mice at multiple frequencies (kHz) in treated and untreated contralateral ears 2 months after AAV-Pou4f3 treatment. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0050] FIG. 16 shows a summary of the auditory brainstem response (ABR) Wave 1 amplitude data for five C57Bl / 6j mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0051] FIG. 17 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for five C57Bl / 6j mice at multiple frequencies (kHz) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001. Values and error bars reflect mean ± SEM.8MF-364562974Docket No.: 336122000640

[0052] FIGs. 18A-18B shows the average auditory brainstem response (ABR) (FIG. 18A) and distortion product otoacoustic emission (DPOAE) (FIG. 18B) thresholds for five C57Bl / 6j mice at multiple frequencies (kHz) in treated and untreated contralateral ears 4 months after AAV-Pou4f3 treatment. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values and error bars reflect mean ± SEM.

[0053] FIG. 19 shows a schematic of a study design for assessing the treatment of permanent threshold shift (PTS) noise induced hearing loss in CBA / CaJ mice with an AAV-Pou4f3 therapy.

[0054] FIG. 20A shows immunofluorescence staining of Pou4f3 in cochleae samples of CBA / CaJ mice 2 months after HOdB, l-20kHz octave band, permanent threshold shift (PTS) noise exposure and AAV-Pou4f3 treatment. Bar=10pm. FIG. 20B shows AAV-Pou4f3 expression level as compared to the endogenous Pou4f3 based on immunostaining intensity of Pou4f3 protein in the hair cells.

[0055] FIG. 21A shows immunofluorescence staining of Myosin 7 A (MYO7A) in the apex, middle (mid), and base of the cochlea of CBA / CaJ mice 3-4 months after HOdB, l-20kHz octave band, permanent threshold shift (PTS) noise exposure and AAV-Pou4f3 treatment. Shown also is the percentage of outer hair cells (OHCs) (FIG. 21B) and inner hair cells (IHCs) (FIG. 21 C) detected in the apex, middle, and base of the cochlea of both the treated and untreated groups. Bar=10pm.

[0056] FIG. 22A shows immunofluorescence staining of C-terminal-binding protein 2 (CtBP2) and Myosin 7 A (MYO7A) in the apex, middle (mid), and base of the cochlea of CBA / CaJ mice 3-4 months after 1 lOdB, 1 -20kHz octave band, permanent threshold shift (PTS) noise exposure and AAV-Pou4f3 treatment. FIG. 22B shows the amount of CtBP2 per inner hair cell (IHC) detected in each region of the cochlea for the treated and untreated groups. Bar=10pm.

[0057] FIG. 23A shows staining of F-Actin by Phalloidin in the apex-middle (Apex-mid) and mid-base regions of the cochlea of CBA / CaJ mice 3-4 months after 1 lOdB, l-20kHz octave band, permanent threshold shift (PTS) noise exposure and AAV-Pou4f3 treatment. FIG. 23B shows the percentage of inner hair cells (IHCs) without stereocilia bundles detected in each region of the cochlea for the treated and untreated groups. Bar=10pm.9MF-364562974Docket No.: 336122000640

[0058] FIG. 24 shows expression levels of multiple inner ear hair cell bundle genes in adult WT mouse after AAV-Pou4f3 treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed unpaired Student’s t-test. Error bar, mean ± SEM.

[0059] FIG. 25 shows a schematic of a study design for assessing the treatment of HOdB sound pressure level (SPL) noise damage in C57Bl / 6j mice with an AAV-Atohl therapy.

[0060] FIG. 26 shows a summary of the auditory brainstem response (ABR) Wave 1 amplitude data for five CBA / CaJ mice at multiple frequencies (kHz) at 100 dB sound pressure level (SPL) in treated and untreated contralateral ears 3 weeks after AAV-Atohl treatment. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01. Values and error bars reflect mean ± SEM.

[0061] FIG. 27 shows the average auditory brainstem response (ABR) Wave 1 amplitudes for five CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 3 weeks after AAV-Atohl treatment for 1 lOdB permanent threshold shift (PTS) noise damage. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01. Values and error bars reflect mean ± SEM.

[0062] FIGs. 28A-28B shows the average auditory brainstem response (ABR) (FIG. 28A) and distortion product otoacoustic emission (DPOAE) (FIG. 28B) thresholds for five CBA / CaJ mice at multiple frequencies (kHz) in treated and untreated contralateral ears 3 weeks after AAV-Atohl treatment for 110 dB permanent threshold shift (PTS) noise damage. N=5 in each group. Statistical tests were two-way Analysis of Variance (ANOVA) with Sidak's multiple comparisons test: *p < 0.05, **p < 0.01. Values and error bars reflect mean ± SEM.DETAILED DESCRIPTION

[0063] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.10MF-364562974Docket No.: 336122000640I. Definitions

[0064] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0065] The term “exogenous” indicates that the nucleic acid or polypeptide is part of, or encoded by, a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.

[0066] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0067] As used herein, the term “increased expression” or “overexpression” means any form of expression that is additional to the expression in an original or source cell that does not contain the modification for modulating a particular gene expression, e.g., a wild-type expression level. Reference herein to “increased expression” or “overexpression” is taken to mean an increase in gene expression and / or, as far as referring to polypeptides, increased polypeptide levels and / or increased polypeptide activity, relative to the level in a cell that does not contain the modification, such as the original source cell prior to the engineering to introduce the modification, such as an unmodified cell or a wild-type cell. In some embodiments, increased expression is measured in an inner ear cell (e.g., a cochlear cell or utricular cell).

[0068] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises 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, such as cosmids, plasmids (e.g., naked11MF-364562974Docket No.: 336122000640 or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0069] As used herein, “Pou4f3” refers to any and all Pou4f3- associated nucleic acid or protein sequences and includes any sequence that is orthologous or homologous to, or has significant sequence similarity to, a Pou4f3 nucleic acid or amino acid sequence derived from any animal including mammals (e.g., humans). The term also includes homologs, orthologs, mutants, variants or fragments thereof. Pou4f3 also includes all other synonyms that may be used to refer to the Pou4f3 gene or the Pou4f3 protein product of the gene (synonyms for the Pou4f3 gene include POU class 4 homeobox 3, BRN3C, DFNA15, DFNA42, and DFNA52). The amino acid sequence of human Pou4f3 appears, for example, in the NCBI database under accession no. NP_002691 (see ncbi.nlm.nih.gov and SEQ ID NO: 4). The nucleic acid sequence of human Pou4f3 appears, for example, in the NCBI database under accession no. NM_002700 (see ncbi.nlm.nih.gov and SEQ ID NO: 3).

[0070] Unless otherwise specified, a “nucleic acid sequence encoding” an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns.

[0071] The term “operably linked” refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.

[0072] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, such as a human subject.12MF-364562974Docket No.: 336122000640

[0073] The term “percent sequence identity” or having “a sequence identity” refers to the degree of identity between any given query sequence and a subject sequence.

[0074] The terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, hinders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.

[0075] The term “polynucleotide” is a chain of nucleotides, also known as a “nucleic acid”. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, and include both naturally occurring and synthetic nucleic acids. As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably throughout the specification and include complementary DNA (cDNA), linear or circular oligomers or polymers of natural and / or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0076] The terms “polypeptide,” “peptide,” and “protein” refer to compounds comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids. Polypeptides include peptides or proteins comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.13MF-364562974Docket No.: 336122000640

[0077] The term “transfected” or “transformed” or “transduced” means to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The transfected / transformed / transduced cell includes the primary subject cell and its progeny.

[0078] To “treat” a disease or disorder as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom associated with the disease or disorder experienced by an individual. Treatment involves an observable beneficial effect of the treating the individual, including a reduction in severity of some or all signs or symptoms associated with the disease or disorder, a slower progression of the disease or disorder, an improvement in the overall health or well-being of the individual, or by other parameters well known in the art that are specific to the particular disease or disorder.

[0079] To “prevent” a disease or disorder, as used herein, refers to inhibiting, delaying, or reducing the severity of the onset of a disease or disorder or reducing the likelihood of developing at least one sign or symptom associated with the disease or disorder in an individual.

[0080] As used herein, “variant” of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[0081] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of vectors include hut are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors,14MF-364562974Docket No.: 336122000640 and the like. The vector can also include a regulatory region. The term “regulatory region” refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and / or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0082] As used herein, the terms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0083] As used here, the term “about” refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0084] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0085] As used herein, the terms “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0086] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.I. Methods of treatment and prevention

[0087] Provided herein are methods of preventing hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating hearing loss in an individual comprising administering to the individual a composition comprising an agent15MF-364562974Docket No.: 336122000640 that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss.

[0088] In some aspects, provided herein are methods of preventing hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects, provided herein are methods of treating hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss.

[0089] Additionally, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating vestibular disorders.

[0090] In some aspects, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects, provided herein are methods of treating a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating vestibular disorders.

[0091] Patients with hearing loss can be identified using standard hearing tests known in the art. In some embodiments, the present disclosure can be used to treat and / or prevent inner ear cell damage (e.g., cochlear cell or utricular cell damage) and associated disorders, such as hearing impairments, deafness, vestibular disorders, tinnitus (see, Kaltenbach et al. (2002) J NEUROPHYSIOL, 88(2). 699-714s, the contents of which is herein incorporated by reference in its entirety), and hyperacusis (Kujawa et al. (2009) J. NEUROSCI. 29(45): 14077- 14085, the contents of which is herein incorporated by reference in its entirety). Also provided herein are methods of preventing and / or treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.16MF-364562974Docket No.: 336122000640

[0092] The individual may be deaf or have hearing loss for any reason, or as a result of any type of event. For example, an individual may be deaf because of a genetic or congenital defect; for example, an individual can be deaf since birth, or can be deaf or hard-of-hearing as a result of a gradual loss of hearing due to a genetic or congenital defect. In another example, an individual can be deaf or hard-of-hearing as a result of a traumatic event, such as a physical trauma to a structure of the ear. For example, prolonged exposures to concerts, airport runways, and construction areas can cause inner ear damage and subsequent hearing loss.

[0093] In some embodiments, the hearing loss is noise-induced hearing loss (e.g., a hearing impairment resulting from exposure to loud sounds or noise, either in a single intense burst or over an extended period). In some embodiments, the hearing loss is occupational hearing loss (e.g., exposure a hearing impairment resulting from exposure to loud sounds or noise while working). In some embodiments, the individual is at risk of developing noise induced hearing loss (e.g., a construction worker or military service member).

[0094] In some embodiments, the methods provided herein treat noise-induced hearing loss in an individual in need thereof, e.g., the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein is administered after exposure to a sudden loud noise or prolonged exposure to loud noises. In some embodiments, the composition is administered to the individual within about 12 hours, about 1 day, about 5 days, about 8 days, 12 days, 14 days, 15 days, about 21 days, about 28 days, or about 30 days after noise exposure (e.g., a sudden loud noise or prolonged exposure to loud noises). In some embodiments, the method recovers hearing in the individual for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, or at least 20 weeks following administration of the composition. Any suitable method known in the art can be used to assess hearing (e.g., Wave I amplitude, ABR threshold, and / or DPOAE threshold).

[0095] In some embodiments, the methods provided herein prevent noise-induced hearing loss in an individual in need thereof, e.g., the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein is administered as a prophylactic against noise damage. In some embodiments, the composition is administered to the individual no more than about 12 hours, about 1 day, about 5 days, or about a week prior to noise exposure (e.g., a sudden loud noise or prolonged exposure to loud noises). In some embodiments, the method17MF-364562974Docket No.: 336122000640 provides protection against noise-induced hearing loss in the individual for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 following administration of the composition.

[0096] In some embodiments, the hearing loss is age-related hearing loss (also referred to as presbycusis). In some embodiments, the age-related hearing loss is characterized by progressive damage and loss of hair cells (e.g., outer hair cells and / or inner hair cells) in the ear of the individual. In some embodiments, the age-related hearing loss is characterized by progressive loss of synapses between spiral ganglion neuron and the inner hair cells (IHCs) in the ear of the individual (e.g., cochlear synaptopathy).

[0097] In some embodiments, the methods provided herein treat age-related hearing loss in an individual in need thereof. In some embodiments, the method recovers hearing in the individual for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, or at least 20 weeks following administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0098] In some embodiments, the methods provided herein prevent age-related hearing loss in an individual in need thereof. In some embodiments, the method provides protection against age-related hearing loss in the individual for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, or at least 16 weeks following administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0099] In humans, the POU4F3 gene is one of the most common autosomal-dominant deafness-associated genes, variants of which cause post-lingual onset, progressive non- syndromic hearing loss (NSHL). In some embodiments, the hearing loss is progressive non- syndromic hearing loss (NSHL). In some embodiments, the methods provided herein treat NSHL. In some embodiments, the methods provided herein prevent NSHL. In some embodiments, the hearing loss is due to a mutation in a POU4F3 gene of the individual. In some embodiments, the individual has at least one mutation (e.g., a point mutation, insertion, deletion, substitution, etc.) in POU4F3 gene. In some embodiments, the individual expresses low levels of a Pou4f3 protein. Several POU4F3 gene variants associated with hearing loss are known in the art, e.g., p.Glnl l3Ter, p.Prol64Arg, p.Leu201fs, p.Gly221fs, p.Leu223Pro,18MF-364562974Docket No.: 336122000640 p.Glu232Lys, p.Leu289Phe, p.Ile295fs, p.Leu311Pro, p.Arg326Lys, p.Ala336fs, whole deletion of POU4F3, and C.120+1G>C {see, e.g., Kitano T et al., POU4F3 mutation screening in Japanese hearing loss patients: Massively parallel DNA sequencing-based analysis identified novel variants associated with autosomal dominant hearing loss. PLoS One. 2017 May 17;12(5):e0177636, the contents of which is herein incorporated by reference in its entirety).

[0100] As demonstrated herein, overexpression of a Pou4f3 protein can recover hearing in C57Bl / 6j mice, which have a genetic mutation in the CDH23 gene. The CDH23 gene encodes the protein Cadherin 23, a glycoprotein necessary for both development and maintenance of the hair bundle and tip links of the hair cells, thus playing an important role in mechanotransduction. Mice homozygous for this mutation show progressive degeneration of stereocilia in the ear and progressive high frequency hearing loss starting at about 3 months of age that eventually results in severe hearing loss at one year of age. Further, humans with CDH23 gene mutations show a broad range of phenotypes from congenital to age-related hearing loss depending on the residual function defined by the CDH23 variants or gene dosage. Accordingly, it is contemplated that increased expression and / or activity of a Pou4f3 protein can increase expression of the CDH23 gene, repair stereocilia, and restore hearing in the ear of an individual, wherein the individual carries at least one copy of a wildtype or a hypomorphic variant of the CDH23 gene. In some embodiments, the hearing loss is progressive hearing loss. In some embodiments, the methods provided herein treat progressive hearing loss. In some embodiments, the methods provided herein prevent progressive hearing loss. In some embodiments, the hearing loss is due to a mutation in a CDH23 gene of the individual. In some embodiments, the individual has at least one mutation (e.g., a point mutation, insertion, deletion, substitution, etc.) in a CDH23 gene. In some embodiments, the individual carries a hypomorphic variant of the CDH23 gene. Several CDH23 gene variants associated with hearing loss are known in the art, e.g., p.P240L, p.R1588W, p.R2029W, and p.E956K see, e.g., Usami SI et al., Variants in CDH23 cause a broad spectrum of hearing loss: from non- syndromic to syndromic hearing loss as well as from congenital to age-related hearing loss. Hum Genet. 2022 Apr;141(3-4):903-914, the contents of which is herein incorporated by reference in its entirety).

[0101] In some embodiments, the hearing loss is progressive hearing loss. In some embodiments, the methods provided herein treat progressive hearing loss. In some embodiments, the methods provided herein prevent progressive hearing loss. In some embodiments, the hearing loss is due to a mutation in a hair cell bundle gene of the individual.19MF-364562974Docket No.: 336122000640In some embodiments, the individual has at least one mutation (e.g., a point mutation, insertion, deletion, substitution, etc.) in a hair cell bundle gene.

[0102] In some embodiments, the hearing loss is sudden hearing loss (e.g., an unexplained and rapid loss of hearing). In some embodiments, the methods provided herein treat sudden hearing loss.

[0103] In some embodiments, the hearing loss is post-lingual hearing loss (e.g., a hearing impairment that develops after the acquisition of speech and language). In some embodiments, the methods provided herein treat post-lingual hearing loss. In some embodiments, the methods provided herein prevent post-lingual hearing loss.

[0104] In some embodiments, the individual exhibits tinnitus (e.g., the perception of sound such as ringing or buzzing that does not have an external source). In some embodiments, the tinnitus is a symptom of the hearing loss in the individual.

[0105] Vestibular disorders (e.g., a condition affecting an individual’s sense of balance) have been shown to be associated with mutations in both the POU4F3 gene and CDH23 gene. For instance, Bm-3c- / - mice showed severe vestibular dysfunction after birth (see, e.g., M. Xiang et al., “Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 17, pp. 9445-9450, 1997 and L. Erkman et al., “Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development,” Nature, vol. 381, no. 6583, pp. 603-606, 1996). In humans, vestibular impairment was identified in individuals with nonsyndromic autosomal dominant deafness 15 (DFNA15) caused by a POU4F3 gene mutation see, e.g., F. J. W. van Drunen et al., “Vestibular impairment in a Dutch DFNA15 family with an L289F mutation in POU4F3,” Audiology and Neurotology, vol. 14, no. 5, pp. 303-307, 2009 and R. J. Pauw et al. “Audiometric characteristics of a Dutch family linked to DFNA15 with a novel mutation (p.L289F) in POU4F3,” Archives of Otolaryngology — Head and Neck Surgery, vol. 134, no. 3, pp. 294-300, 2008). Accordingly, it is contemplated that increased expression and / or activity of a Pou4f3 protein can treat and / or prevent a vestibular disorder in an individual. In some embodiments, the individual with the vestibular disorder exhibits dizziness and vertigo. In some embodiments, the vestibular disorder is associated with the hearing loss in the individual. In some embodiments, the vestibular disorder is associated with progressive hearing loss. In some embodiments, the vestibular disorder is associated with age-related hearing loss.20MF-364562974Docket No.: 336122000640

[0106] Where appropriate, before and / or following treatment, vestibular testing can be performed on the individual. In some embodiments, vestibular testing is performed on the individual to assess whether the vestibular disorder in the individual has improved. Vestibular tests are known in the art and include but are not limited to: electronystagmography (ENG), head thrust test, balance platform test, videonystagmography (VNG), rotational testing, video head impulse testing (VHIT), vestibular short-latency evoked potential (VsEP), vestibular evoked myogenic potential (VEMP), and computerized dynamic posturography (CDP).

[0107] In some embodiments, the vestibular test is an electronystagmography (ENG) test, wherein a warm or cool stimulus (e.g., air or water) is placed in the individual’s ear canal and the caloric response of stimulus to the lateral semicircular canal is measured by eye motion.

[0108] In some embodiments, the vestibular test is a head thrust test which can involve quickly turning the individual’s head in a direction to localize one set of semicircular canals, one from each ear. In some embodiments, the paired response of the lateral canals is tested with the head tilted forward 30 degrees to make the lateral canals horizontal. The individual is instructed to fix their gaze on a point straight ahead. When the examiner conducting the test quickly turns the head of the individual toward the ear with a weak vestibular response, the eyes move from the target and then bounce back to the target with a catch-up saccade.

[0109] In some embodiments, the vestibular test involves a balance platform used to measure an individual’s sway when visual or proprioceptive clues are removed.

[0110] In some embodiments, the methods described herein improve the vestibular disorder of an individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 40%, at least 60%, at least 80, or at least 90% relative to the vestibular disorder prior to administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0111] In some embodiments, the individual exhibits cochlear synaptopathy (e.g., loss of synapses between spiral ganglion neuron and the inner hair cells (IHCs) in the ear of the individual; also referred to as hidden hearing loss).

[0112] In some embodiments, the hearing loss effects one ear of the individual. In some embodiments, the hearing effects both ears of the individual. In some embodiments, the individual is human.

[0113] The number of doses of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein and duration of treatment can be varied to achieve a desired result. In some embodiments, the composition is administered as a single21MF-364562974Docket No.: 336122000640 dose. In some embodiments, the composition is administered in multiple doses, e.g., at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 doses. In some embodiments, the duration of treatment is one to six days, one week, two weeks, three weeks, one month, three months, six months, nine months, one year, two years or more.

[0114] In some embodiments, the methods described herein improve the hearing of an individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 40%, at least 60%, at least 80, or at least 90% relative to the hearing prior to administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

[0115] In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein results in increased expression (i.e., overexpression) of the Pou4f3 protein in the ear of the individual (e.g., a cochlear cell or utricular cell in the ear of the individual). In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual by at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold as compared to the level of the Pou4f3 protein in the cochlear cell in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual by about 2-fold as compared to the level of the Pou4f3 protein in the cochlear cell in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual as compared to the level of the Pou4f3 protein in a cochlear cell in the ear of an individual who did not receive the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual by at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold as compared to the level of the Pou4f3 protein in a cochlear cell in the ear of an individual who did not receive the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual by about 1.25-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, or about 5-fold as compared to the level of the Pou4f3 protein in a cochlear cell in the ear of an individual who did not receive the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 protein in a cochlear cell in the ear of the individual by about 2-fold as compared to the level of the Pou4f3 protein in a cochlear cell in the ear of an individual who did not receive the composition. Any suitable22MF-364562974Docket No.: 336122000640 method can be used to measure Pou4f3 protein expression (e.g., flow cytometry, ELISA, or western blot). In some embodiments, Pou4f3 protein expression levels are measured in an inner ear tissue explant sample (e.g., a cochlear tissue or utricular tissue explant sample).

[0116] In some embodiments, administration of the composition results in increased expression (i.e., overexpression) of the Pou4f3 protein in the ear of the individual (e.g., a cochlear cell or utricular cell in the ear of the individual), wherein the composition comprises a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, Pou4f3 protein expression levels are predicted based on pharmacokinetic (PK) studies in an animal model. In animal PK studies, Pou4f3 protein levels in inner ear cells can be correlated with the viral vector (e.g., AAV vector) titer and injection volume.

[0117] In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein results in increased levels of Pou4f3 mRNA in the ear of the individual (e.g., a cochlear cell or utricular cell in the ear of the individual). In some embodiments, prior to administration of the composition, the individual expresses a low level of Pou4f3 mRNA in a cochlear cell in the individual’s ear. In some embodiments, administration of the composition increases the level of Pou4f3 mRNA in a cochlear cell in the ear of the individual as compared to the level of Pou4f3 mRNA in the cochlear cell in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the level of Pou4f3 mRNA in a cochlear cell in the ear of the individual as compared to the level of Pou4f3 mRNA in a cochlear cell in the ear of an individual who did not receive the composition. Any suitable method can be used to measure Pou4f3 mRNA levels (e.g., Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT- PCR)). In some embodiments, Pou4f3 mRNA expression levels are measured in an inner ear tissue explant sample (e.g., a cochlear tissue or utricular tissue explant sample).

[0118] In some embodiments, administration of the composition results in increased levels of Pou4f3 mRNA in the ear of the individual (e.g., a cochlear cell or utricular cell in the ear of the individual), wherein the composition comprises a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, Pou4f3 mRNA levels are predicted based on pharmacokinetic (PK) studies in an animal model. In animal PK studies, Pou4f3 mRNA levels in inner ear cells can be correlated with the viral vector (e.g., AAV vector) titer and injection volume.23MF-364562974Docket No.: 336122000640

[0119] In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein results in increased biological activity of the Pou4f3 protein in the ear of the individual (e.g., a cochlear cell or utricular cell in the ear of the individual). In some embodiments, the increased biological activity is the upregulation of one or more inner ear hair cell bundle genes. In some embodiments, administration of the composition increases the expression level (e.g., mRNA level) of a hair cell bundle gene in the ear of the individual by at least 1.25-fold, at least 1.5-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, or at least 12-fold as compared to the expression level of the hair cell bundle gene in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the expression level of a hair cell bundle gene in the ear of the individual by about 1.25-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 10-fold, or about 12-fold as compared to the expression level of the hair cell bundle gene in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the expression level of one or more hair cell bundle genes in the ear of the individual as compared to the expression level of the one or more hair cell bundle genes in the ear of an individual who did not receive the composition. In some embodiments, administration of the composition increases the expression level of a hair cell bundle gene in the ear of the individual by at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, or at least 12-fold as compared to the expression level of the hair cell bundle gene in the ear of an individual who did not receive the composition. In some embodiments, administration of the composition increases the expression level of a hair cell bundle gene in the ear of the individual by about 1.25-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 10-fold, or about 12-fold as compared to the expression level of the hair cell bundle gene in the ear of an individual who did not receive the composition. In some embodiments, the hair cell bundle gene is selected from the group consisting of Atp8bl, Myo7a, Espn, Cdh23, Pcdhl5, Tmcl, Ptprq, Strc, Ckb, Myol5, Tmie, Atp2b2, Ush2a. In some embodiments, the hair cell bundle gene is Atp8bl, which encodes the protein phospholipid-transporting ATPase IC. In some embodiments, the hair cell bundle gene is Myo7a, which encodes the protein myosin 7a. In some embodiments, the hair cell bundle gene is Espn, which encodes the protein espin. In some embodiments, the hair cell bundle gene24MF-364562974Docket No.: 336122000640 is Cdh23, which encodes the protein cadherin-23. In some embodiments, the hair cell bundle gene is Pcdhl5, which encodes the protein protocadherin-15. In some embodiments, the hair cell bundle gene is Tmcl, which encodes the protein transmembrane channel-like protein 1. In some embodiments, the hair cell bundle gene is Ptprq, which encodes protein tyrosine phosphatase receptor type Q. In some embodiments, the hair cell bundle gene is Sire. which encodes the protein stereocilin. In some embodiments, the hair cell bundle gene is Ckb, which encodes the protein creatine kinase B. In some embodiments, the hair cell bundle gene is Myol5, which encodes the protein myosin 15. In some embodiments, the hair cell bundle gene is Tmie, which encodes the protein transmembrane inner ear expressed protein. In some embodiments, the hair cell bundle gene is Atp2b2, which encodes the protein plasma membrane calcium-transporting ATPase 2. In some embodiments, the hair cell bundle gene is Ush2a, which encodes the protein usherin. Any suitable method can be used to measure hair cell bundle gene expression levels (e.g., mRNA levels) including, but not limited to, Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR). In some embodiments, hair cell bundle gene expression levels are measured in an inner ear tissue explant sample (e.g., a cochlear tissue or utricular tissue explant sample).

[0120] Where appropriate, before and / or following treatment, the hearing of the individual can be assessed. In some embodiments, the hearing of the individual is assessed to determine whether the hearing has improved. Methods for measuring hearing are well-known and include, but are not limited to, pure tone audiometry, air conduction, auditory brainstem response (ABR) and bone conduction tests. These exams can measure the limits of loudness (intensity) and pitch (frequency) that an individual can hear. Hearing tests in humans include behavioral observation audiometry (for infants to seven months), visual reinforcement orientation audiometry (for children 7 months to 3 years) and play audiometry for children older than 3 years. Oto-acoustic emission testing can be used to test the functioning of the cochlea hair cells, and electro-cochleography provides information about the functioning of the cochlea and the first part of the nerve pathway to the brain.

[0121] In some embodiments, hearing of the individual is assessed by auditory brainstem response (ABR). ABR is a measurement of auditory pathway function from the auditory nerve to the mesencephalon (Young A. et al., Auditory Brainstem Response. [Updated 2023 Jan 12], In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan., the contents of which is herein incorporated by reference in its entirety). In some embodiments, ABR threshold25MF-364562974Docket No.: 336122000640 provides a metric for overt hearing loss. Any suitable method known in the art can be used for recording ABR. In some embodiments, the method further comprises determining the ABR threshold (e.g., a minimum level of sound intensity that elicits a detectable ABR) of the individual at one or more frequencies (e.g., about 5, about 8, about 11, about 16, about 22, and about 32 kHz). In some embodiments, the method comprises determining the ABR threshold of the individual at one or more frequencies before and after the individual has received the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the individual does not exhibit a substantial change in ABR threshold (e.g., the ABR threshold does not increase) at one or more frequencies as compared to the ABR threshold of the individual at the one or more frequences prior to administration of the composition (e.g., such that administration of the composition prevents hearing loss in an individual). In some embodiments, administration of the composition results in a decrease in the ABR threshold of the individual at one or more frequencies as compared to the ABR threshold of the individual at the one or more frequencies prior to administration of the composition (e.g., such that administration of the composition improves hearing and / or treats hearing loss in an individual). In some embodiments, administration of the composition results in a decrease in the ABR threshold of the individual at one or more frequencies as compared to the ABR threshold of an individual at the one or more frequencies who did not receive the composition.

[0122] In some embodiments, hearing of the individual is assessed by determining the Wave I amplitude of the individual. Wave 1 amplitude represents the summed activity of the auditory nerve fibers contacting inner hair cells (IHCs) and can be used to assess the individual for potential cochlear synaptopathy. Any suitable method known in the art can be used for determining Wave I amplitude. In some embodiments, the method further comprises determining the Wave I amplitude of the individual at one or more frequencies (e.g., about 5, about 8, about 11, about 16, about 22, and about 32 kHz). In some embodiments, the method further comprises determining the Wave I amplitude of the individual at one or more frequencies before and after the individual has received the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the individual does not exhibit a substantial change in Wave I amplitude (e.g., the Wave I amplitude does not decrease) at one or more frequencies as compared to the Wave I amplitude of the individual at the one or more frequencies prior to administration of the composition (e.g., such that administration of the composition prevents hearing loss in an individual). In some26MF-364562974Docket No.: 336122000640 embodiments, administration of the composition results in an increase in the Wave I amplitude of the individual at one or more frequencies as compared to the Wave I amplitude of the individual at the one or more frequencies prior to administration of the composition (e.g., such that administration of the composition improves hearing and / or treats hearing loss in an individual). In some embodiments, administration of the composition results in an increase in the Wave I amplitude of the individual at one or more frequencies as compared to the Wave I amplitude of an individual who did not receive the composition at the one or more frequencies.

[0123] In some embodiments, hearing of the individual is assessed by distortion product otoacoustic emission (DPOAE). The DPOAE read-out depends on the biological motors in outer hair cells (OHCs) which amplify sound-evoked cochlear vibration. DPOAE threshold can thus provide a measure of OHC damage. Any suitable method known in the art can be used for determining DPOAE threshold. In some embodiments, the method further comprises determining the distortion product otoacoustic emission (DPOAE) threshold of the individual at one or more frequencies (e.g., about 5, about 8, about 11, about 16, about 22, and about 32 kHz). In some embodiments, the method further comprises determining the distortion product otoacoustic emission (DPOAE) threshold of the individual at one or more frequencies before and after the individual has received the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the individual does not exhibit a substantial change in DPOAE threshold (e.g., the DPOAE threshold does not increase) at one or more frequencies as compared to the DPOAE threshold of the individual at the one or more frequencies prior to administration of the composition (e.g., such that administration of the composition prevents hearing loss in an individual). In some embodiments, administration of the composition results in a decrease in the DPOAE threshold of the individual at one or more frequencies as compared to the DPOAE threshold of the individual at the one or more frequencies prior to administration of the composition (e.g., such that administration of the composition improves hearing and / or prevents hearing loss in an individual). In some embodiments, administration of the composition results in a decrease in the DPOAE threshold of the individual at one or more frequencies as compared to the DPOAE threshold of an individual who did not receive the composition at the one or more frequencies.

[0124] It is contemplated that increasing Pou4f3 expression and / or activity in an individual repairs the outer hair cells and / or inner hair cells in the ear of the individual (e.g., after noise exposure or due to aging). In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein reduces and / or27MF-364562974Docket No.: 336122000640 prevents the loss of the outer hair cells and / or inner hair cells in the ear of the individual. In some embodiments, administration of the composition reduces the loss of the outer hair cells and / or inner hair cells in the ear of the individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of the composition reduces the loss of the outer hair cells and / or inner hair cells in the ear of the individual by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, administration of the composition reduces and / or prevents the loss of the outer hair cells and / or inner hair cells in the ear of the individual as compared to the loss of the outer hair cells and / or inner hair cells in the ear of an individual who did not receive the composition.

[0125] It is contemplated that increasing Pou4f3 expression and / or activity in an individual repairs the synapses between inner hair cells and spiral ganglion neurons in the ear of the individual (e.g., after noise exposure or due to aging). In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein reduces and / or prevents the loss of the synapses between inner hair cells and spiral ganglion neurons in the ear of the individual. In some embodiments, administration of the composition reduces the loss of the synapses between inner hair cells and spiral ganglion neurons in the ear of the individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of the composition reduces the loss of the synapses between inner hair cells and spiral ganglion neurons in the ear of the individual by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, administration of the composition reduces and / or prevents the loss of the synapses between inner hair cells and spiral ganglion neurons in the ear of the individual as compared to the loss of the synapses between inner hair cells and spiral ganglion neurons in the ear of an individual who did not receive the composition.28MF-364562974Docket No.: 336122000640

[0126] It is contemplated that increasing Pou4f3 expression and / or activity in an individual repairs stereocilia of hair cells in the ear of the individual (e.g., after noise exposure or due to aging). In some embodiments, administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein reduces and / or prevents the loss of stereocilia in the ear of the individual. In some embodiments, administration of the composition reduces the loss of stereocilia in the ear of the individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of the composition reduces the loss of stereocilia in the ear of the individual by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, administration of the composition reduces and / or prevents the loss of stereocilia in the ear of the individual as compared to the loss of stereocilia in the ear of an individual who did not receive the composition.

[0127] Cell markers can also be used to determine whether an inner ear cell, e.g., a cochlear cell or utricular cell, has been repaired following administration of the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Exemplary markers indicative of hair cells include, but are not limited to, Myo7a, Myo6, Prestin, Lhx3, Dner, espin, parvalbumin, F-actin, and calretinin. Exemplary markers indicative of supporting cells include, but are not limited to, Sox2, SlOOal, Proxl, Rps6, and Jagl. In addition, neuronal markers, e.g., acetylated tubulin, neurofilament and C-terminal-binding protein 2 (CtBP2), can be used to detect neuronal structure, to determine whether hair cells are in contact with neurons. The presence of neuronal markers adjacent to or in contact with hair cells suggests that hair cells have been repaired and / or preserved and possess synapses with neurons (e.g., spiral ganglion neurons).Inner Ear Cells

[0128] In some embodiments, the composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein is administered to the inner ear of the individual. In some embodiments, administration of the composition results in an increased level of a Pou4f3 molecule (e.g., a Pou4f3 protein or Pou4f3 mRNA) in an inner ear cell of the individual (e.g., a cochlear cell or utricular cell).29MF-364562974Docket No.: 336122000640

[0129] In some embodiments, the composition is administered to a cochlear cell in the ear of the individual. In some embodiments, administration of the composition results in an increased level of a Pou4f3 molecule (e.g., a Pou4f3 protein or Pou4f3 mRNA) in a cochlear cell in the ear of the individual. In some embodiments, prior to administration of the composition, the individual expresses a low level of a Pou4f3 molecule. In some embodiments, administration of the composition increases the level of the Pou4f3 molecule in a cochlear cell in the ear of the individual as compared to the level of the Pou4f3 molecule in the cochlear cell in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 molecule in a cochlear cell in the ear of the individual as compared to the level of the Pou4f3 molecule in a cochlear cell in the ear of an individual who did not receive the composition. In some embodiments, the cochlear cell is selected from the group consisting of a stria vascularis cell, a hair cell, a supporting cell, and a spiral ganglion neuron. In some embodiments, the cochlear cell is an inner hair cell. In some embodiments, the cochlear cell is an outer hair cell.

[0130] In some embodiments, the composition is administered to a utricular cell in the ear of the individual. In some embodiments, administration of the composition results in an increased level of a Pou4f3 molecule (e.g., a Pou4f3 protein or Pou4f3 mRNA) in a utricular cell in the ear of the individual. In some embodiments, prior to administration of the composition, the individual expresses a low level of a Pou4f3 molecule. In some embodiments, administration of the composition increases the level of the Pou4f3 molecule in a utricular cell in the ear of the individual as compared to the level of the Pou4f3 molecule in the utricular cell in the ear of the individual prior to administration of the composition. In some embodiments, administration of the composition increases the level of the Pou4f3 molecule in a utricular cell in the ear of the individual as compared to the level of the Pou4f3 molecule in a utricular cell in the ear of an individual who did not receive the composition. In some embodiments, the utricular cell is a vestibular hair cell.

[0131] Sensory epithelia of the inner ear contain two major cell types: hair cells and supporting cells. (G. Wan et al., Semin Cell Dev Biol. 2013 May; 24(5): 448-459). Hair cells convert the energy in sound and head movements into neurophysiological signals that are relayed to the brainstem. In mammals, six sensory organs contain hair-cell epithelia. In the cochlear organ, which is specialized for hearing, hair cells reside within the organ of Corti, atop the basilar membrane, which vibrates in response to sound waves. Similarly, each of the five vestibular organs (the utricle, the saccule, and the three canal organs) contains sensory30MF-364562974Docket No.: 336122000640 epithelia with hair cells that are activated by head movements and gravitational force. Hair cells are innervated by neurons whose cell bodies sit outside the sensory epithelium, either in a sensory ganglion within the temporal bone (afferent neurons) or in the hindbrain (efferent neurons).

[0132] There are two types of hair cells in the cochlea: outer and inner hair cells. Outer hair cells are distal from the spiral limbus, and generally there are three to five rows of hair cells that run the length of the cochlear duct (about 20,000 in number in humans). Inner hair cells are proximal to the spiral limbus. There is only one row of inner hair cells that run the length of the cochlear duct (about 3500 in number in humans).

[0133] In some embodiments, the cochlear cell is a supporting cell. The development, function, and maintenance of inner ear sensory epithelia are heavily dependent upon the supporting cells, which are non-sensory cells that reside between hair cells (Wan et al., supra). Unlike hair cells, which contact only the lumenal surface of the epithelium, supporting cells span the entire depth of the epithelium, from the basal lamina to the lumen. Supporting cells are linked to each other and to hair cells by tight and adherens junctions; and they communicate directly with other supporting cells by gap junctions. Within the mature sensory epithelia, supporting cells share many morphological and molecular features. For instance, supporting cells in mammalian auditory and / or vestibular epithelia express the following genes at the protein and / or transcript level: Sox2, Sox9, SoxlO, Jaggedl, S100a, and p27kipl. However, consistent with the large range of supporting cell functions, supporting cells in a given sensory epithelium show variation with respect to their shapes and molecular profiles. This is most pronounced in the mammalian organ of Corti, which has the greatest degree of supporting cell heterogeneity. Five different types of supporting cells are organized in rows along the organ's length. From the outer edge to the inner edge of the organ, they are: 1) Hensen’s cells, 2) Deiters’ cells, 3) pillar cells; 4) inner phalangeal cells; and 5) border cells. These supporting cells have distinct morphologies. Hensen's cells are cuboidal or slightly oblong. Inner phalangeal cells and border cells are columnar. The remaining cells — Deiters' and pillar cells — are architecturally exquisite cells, with a strong cytoskeleton, elongated processes, and large structural demands. For instance, the inner and outer pillar cells must maintain the structure of the tunnel of Corti, despite pressure from cells located on either side of the tunnel, during acoustic stimulation.

[0134] In some embodiments, the cochlear cell is a stria vascularis cell. The stria vascularis (SV) is a heterogenous tissue located in the lateral wall of the cochlear duct. It is a stratified31MF-364562974Docket No.: 336122000640 epithelium consisting of three major cell types (marginal, intermediate and basal cells) (Gu, S., Olszewski, R., Taukulis, I. et al. Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea. Sci Rep 10, 18100 (2020), the contents of which is herein incorporated by reference in its entirety).

[0135] In some embodiments, the cochlear cell is a spiral ganglion neuron. The spiral ganglion, also known as the cochlear ganglion, is an important structure in the auditory system, playing a role in hearing. It consists of a collection of nerve cells (spiral ganglion neurone) that transmit auditory information from the inner ear to the brain.II. Compositions for increasing Pou4f3 expression and / or activity

[0136] Provided herein are methods of preventing hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss in an individual.

[0137] In some aspects, provided herein are methods of preventing hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects, provided herein are methods of treating hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss.

[0138] Additionally, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating a vestibular disorder in an individual.

[0139] In some aspects, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects,32MF-364562974Docket No.: 336122000640 provided herein are methods of treating a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating vestibular disorders.

[0140] Pou4f3 is a transcription factor and is highly expressed in cochlear and vestibular hair cells in the inner ear, where it is involved in the maturation, differentiation, and survival of cochlear and vestibular hair cells. In humans, the POU4F3 gene is one of the most common autosomal-dominant deafness-associated genes, variants of which cause post-lingual onset, progressive non-syndromic hearing loss (NSHL) (Lee, SY., et al. Ramifications of POU4F3 variants associated with autosomal dominant hearing loss in various molecular aspects. Sci Rep 13, 12584 (2023)).

[0141] In some embodiments, the agent is a nucleic acid (e.g., DNA or RNA). In some embodiments, the agent comprises a nucleic acid sequence encoding the Pou4f3 protein. In some embodiments, increasing expression (i.e., overexpression) and / or activity of the Pou4f3 protein is mediated by introducing into the cell an exogenous nucleic acid encoding the Pou4f3 protein to be overexpressed. In some embodiments, the exogenous nucleic acid is a recombinant nucleic acid. Techniques for generating recombinant nucleic acids are well- known in the art. In some embodiments, an exogenous nucleic acid sequence encoding a Pou4f3 protein comprises a codon-optimized nucleic acid sequence.

[0142] Exemplary human and mouse Pou4f3 nucleic acid sequences are provided in Table 1. The human Pou4f3 cDNA sequence (SEQ ID NO: 3) is 91% homologous to the mouse Pou4f3 cDNA (SEQ ID NO: 1).

[0143] In some embodiments, the Pou4f3 protein is a mouse Pou4f3 protein. In some embodiments, the nucleic acid sequence encoding the mouse Pou4f3 protein comprises a nucleic acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding the mouse Pou4f3 protein comprises a nucleic acid sequence with at least 90% identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding the mouse Pou4f3 protein comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the mouse Pou4f3 protein comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments,33MF-364562974Docket No.: 336122000640 the mouse Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the mouse Pou4f3 protein comprises the amino acid sequence of SEQ ID NO: 2.

[0144] In some embodiments, the Pou4f3 protein is a human Pou4f3 protein. In some embodiments, the nucleic acid sequence encoding the human Pou4f3 protein comprises a nucleic acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encoding the human Pou4f3 protein comprises a nucleic acid sequence with at least 90% identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encoding the human Pou4f3 protein comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the human Pou4f3 protein comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human Pou4f3 protein comprises the amino acid sequence of SEQ ID NO: 4.Table 1 Pou4f3 Sequences34MF-364562974Docket No.: 33612200064035MF-364562974Docket No.: 336122000640

[0145] In some embodiments, a nucleic acid sequence encoding a Pou4f3 protein is an unmodified wild-type sequence. Alternatively, a nucleic acid sequence encoding a Pou4f3 protein can be modified using standard techniques. For example, in some embodiments, a nucleic acid sequence encoding a Pou4f3 protein can be modified or mutated, e.g., to increase the stability of the nucleic acid sequence or resulting protein. In some embodiments, proteins resulting from such altered nucleic acids sequences retain the biological activity of wild type Pou4f3 protein. In some embodiments, a nucleic acid sequence encoding a Pou4f3 protein is altered to increase nuclear translocation of the resulting protein. In some embodiments, a nucleic acid sequence encoding a Pou4f3 protein is modified using standard molecular biological techniques to include an additional nucleic acid sequence that can encode one or more of, e.g., detectable polypeptides, signal peptides, and protease cleavage sites.

[0146] In some embodiments, the nucleic acids encoding a Pou4f3 protein is operably linked to one or more regulatory nucleotide sequences in an expression construct. Typically, regulatory nucleotide sequences are selected for the target cell (e.g., cochlear cell or utricular cells) and the individual to be treated. Several types of expression vectors and suitable regulatory sequences are known in the art for a variety of target cells. Examples of regulatory nucleotide sequences include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, translational start and termination sequences, transcriptional start and termination sequences, and enhancer or activator sequences. Constitutive, inducible, and tissue-specific promoters known in the art are contemplated. In some embodiments, the promoter is a naturally occurring promoter. In some embodiments, the promoter is a hybrid promoter that combines elements of more than one promoter. In some36MF-364562974Docket No.: 336122000640 embodiments, an expression construct is presented in a cell on an episome (e.g., a plasmid). In some embodiments, the expression construct is inserted in a chromosome. In some embodiments, the expression vector comprises a nucleotide sequence encoding a Pou4f3 protein operably linked to at least one regulatory sequence (e.g., promoters, enhancers, and other expression control elements).

[0147] In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a promoter sequence for expression of the protein. In some embodiments, the promoter sequence is selected from the group consisting of a constitutive promoter sequence, an inducible promoter sequence, and a tissue- specific promoter sequence.

[0148] Any suitable promoter known in the art can be operably linked to the nucleic acid sequence encoding the Pou4f3 protein. Examples of suitable promoters include, but are not limited to, a human cytomegalovirus (CMV) promoter, a mammalian elongation factor la (EFla) promoter, a chicken P-actin / CMV hybrid (CAG) promoter, a mammalian promoter from phospholycerate kinase gene (PGK), a tetracycline response element (TRE) promoter, a simian virus 40 (SV40) promoter, a ubiquitin C (UbC) promoter, a retroviral long terminal repeat (LTR) promoter, CBh promoter, and an adenovirus major late promoter (MLP).

[0149] In some embodiments, the promoter sequence is a constitutive promoter sequence. In some embodiments, the constitutive promoter sequence is selected from the group consisting of a human cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, a simian virus 40 (SV40) promoter sequence, and a mammalian elongation factor la (EFla) promoter sequence. In some embodiments, the constitutive promoter sequence is a CMV promoter sequence.

[0150] In some embodiments, the promoter sequence is a tissue-specific promoter sequence. In some embodiments, the tissue- specific promoter sequence is selected from the group consisting of a stria vascularis cell- specific promoter sequence, a hair cell- specific promoter sequence, a supporting cell-specific promoter sequence, and a spiral ganglion neuron-specific promoter sequence.

[0151] In some embodiments, the tissue- specific promoter sequence is a hair cell-specific promoter sequence. Examples of hair cell-specific promoters, include, without limitation: a myosin VIIA promoter, a myosin VI promoter, a myosin 15 promoter, a stereocilin (Strc) promoter, a Otoferlin promoter, a Slc26A5 (Prestin) promoter, and a Pou4f3 promoter.

[0152] In some embodiments, the tissue-specific promoter sequence is a spiral ganglion neuron-specific promoter sequence. Examples of spiral ganglion neuron-specific promoters,37MF-364562974Docket No.: 336122000640 include, without limitation: an ephrinB2, ephrinB3, trkB, trkc, GATA3, BF1, FGF10, FGF3, CSP, GFAP, or Islet 1 promoter.

[0153] In some embodiments, the tissue- specific promoter sequence is a supporting cellspecific promoter sequence. Examples of supporting cell-specific promoters, include, without limitation: a glial fibrillary acidic protein (GFAP) promoter, an excitatory amino acid transporter- 1 (EAAT1) promoter, a glutamate transporter (GLAST) promoter or a murine cytomegalovirus (mCMV) promoter.

[0154] The process of introducing a nucleic acid into cells can be achieved by any suitable technique, e.g., calcium phosphate or lipid- mediated transfection, electroporation, fusogens, extracellular vesicles, and transduction or infection using a viral vector. In some embodiments, the nucleic acid is introduced into a cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the nucleic acid is introduced into a cell via fusogen-mediated delivery. In some embodiments, the nucleic acid is delivered to the target cell in a vector, e.g., DNA vectors, RNA vectors, plasmids, viral vectors and particles. In some embodiments, lipid nanoparticles are used to deliver the nucleic acid to a cell.

[0155] In some embodiments, the agent is a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. Typically, a viral vector is capable of mediating delivery of a nucleic acid to a host cell and can comprise three main components as follows: (1) the protein capsid and / or envelope which encapsidates the genetic payload, and can define the vector’s tissue or cell tropism and antigen recognition; (2) the transgene of interest, which when expressed in cells, serves to confer a desired effect; and (3) the “regulatory cassette,” the combined enhancer / promoter / auxiliary elements that controls stable or transient somatic expression of the transgene as an episome or as a chromosomal integrant (Bulcha, J.T. et al. Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther 6, 53 (2021), the contents of which is herein incorporated by reference in its entirety). Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. Methods for making and using viral vectors are well known and described in the art.

[0156] In some embodiments, the viral vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vesicular stomatitis virus (VSV) vector, a herpes simplex virus (HSV) vector, a vaccinia virus vector, a38MF-364562974Docket No.: 336122000640 pox virus vector, an influenza virus vector, a respiratory syncytial virus vector, a parainfluenza virus vector, a foamy virus vector, oncolytic viruses, and a retrovirus vector. In some embodiments, the viral vector is an adeno-associated virus type 2 (AAV2) vector.

[0157] In some embodiments, the viral vector is a lentiviral vector. Lentiviruses constitute a genus of the retroviridae family. Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Bioiecknol, 1998, 9: 457-463). Lentiviral vectors can also permit long-term transgene expression. Further, lentiviral vectors generally elicit relatively weak immune responses. Examples of lentiviruses that can be used for viral vectors include, but are not limited to, HIV-1 and HIV -2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia, virus, visna- maedi and caprine arthritis encephalitis virus (CAEV). In some embodiments, a lentiviral vector comprises a central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer.

[0158] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. The AAV vector can comprise any known serotype capsids or combinations of serotype capsids, e.g., AAV1, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12 and AAVrhlO.

[0159] It is contemplated that the methods provided herein increase Pou4f3 expression and activity in an individual which repairs the outer hair cells and / or inner hair cells in the ear of the individual (e.g., after noise exposure or due to aging). In some embodiments, the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a promoter sequence (e.g., a constitutive promoter sequence, an inducible promoter sequence, or a tissue-specific promoter sequence). In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4.

[0160] In some embodiments, the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f339MF-364562974Docket No.: 336122000640 protein. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a tissue-specific promoter sequence. In some embodiments, the tissue- specific promoter sequence is selected from the group consisting of a stria vascularis cell-specific promoter sequence, a hair cell-specific promoter sequence, a supporting cell-specific promoter sequence, and a spiral ganglion neuron-specific promoter sequence. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

[0161] In some embodiments, the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a constitutive promoter sequence. In some embodiments, the constitutive promoter sequence is selected from the group consisting of a human cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, a simian virus 40 (SV40) promoter sequence, and a mammalian elongation factor la (EFla) promoter sequence. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

[0162] In some embodiments, the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the viral vector is an adeno-associated virus type 2 (AAV2) vector. In some embodiments, the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a human cytomegalovirus (CMV) promoter sequence. In some embodiments, the Pou4f3 protein encoded by the nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 4.

[0163] In some embodiments, the method provided herein treats noise-induced hearing loss in an individual in need thereof, e.g., after exposure to a sudden loud noise or prolonged exposure to loud noises, wherein the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. Any suitable method known in the art can be used to assess hearing (e.g., Wave I amplitude, ABR threshold, and / or DPOAE threshold).

[0164] In some embodiments, the method provided herein prevents noise-induced hearing loss in an individual in need thereof, e.g., as a prophylactic against noise damage, wherein the40MF-364562974Docket No.: 336122000640 method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the method provides protection against noise-induced hearing loss in the individual for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 following administration of the composition.

[0165] In some embodiments, the method provided herein treats age-related hearing loss in an individual in need thereof, wherein the method comprises administering to the ear of the individual a composition comprising a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein.

[0166] In some embodiments, the agent is a Pou4f3 protein. In some embodiments, increasing Pou4f3 expression and / or activity is mediated by administering to the individual an exogenous Pou4f3 protein.

[0167] A Pou4f3 protein can be modified to enhance uptake of the protein into target cells (e.g., cochlear cells or utricular cells). In some embodiments, the Pou4f3 protein is mutated to include amino acid sequences that enhance uptake of the protein into a target cell. In some embodiments, a Pou4f3 protein is altered or mutated to increase the stability and / or activity of the protein (e.g., point mutants). In some embodiments, a Pou4f3 protein is altered to increase nuclear translocation of the polypeptide. In some embodiments, an altered Pou4f3 protein or biologically active fragments thereof retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biological activity of full-length, wild type protein.

[0168] Exemplary human and mouse Pou4f3 amino acid sequences are provided in Table 1. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Pou4f3 protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Pou4f3 protein comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Pou4f3 protein comprises the amino acid sequence of SEQ ID NO: 4.41MF-364562974Docket No.: 336122000640

[0169] Sequence identity may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, which are used to perform sequence alignments and then calculate sequence identity. Exemplary software programs available from the National Center for Biotechnology Information (NCBI) on the website ncbi.nlm.nih.gov include blastp, blastn, blastx, tblastn and tblastx. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (e.g., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are used at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. Sci. USA 89:10915-10919). In some embodiments, the percent identity can be determined using the default parameters of blastp, version 2.2.26 available from the NCBI.

[0170] Pou4f3 levels (e.g., protein levels) and / or activity (e.g., biological activity) in target cells (e.g., cochlear cells or utricular cells) can be assessed using any suitable method known in the art, e.g., Western Blotting, in situ hybridization, reverse transcriptase polymerase chain reaction, immunocytochemistry, viral titer detection, and genetic reporter assays. Increases in Pou4f3 levels (e.g., protein levels) and / or activity (e.g., biological activity) in target cells, e.g., from contacting a sample with an agent contemplated to increase Pou4f3 levels and / or activity, can be assessed by comparing Pou4f3 levels and / or activity in a first cell sample or a standard with Pou4f3 levels and / or activity in a second cell sample.

[0171] In some embodiments, Pouf43 levels (e.g., protein) and / or activity can be increased using compounds (e.g., a small molecule or small activating RNAs) that target Pou4f3 or one or more components of the Pou4f3 pathway. Exemplary activators of Pou4f3 include, but are not limited to, aldehyde dehydrogenase inhibitors (e.g., 4-diethylaminobenzaldehyde (DEAB)), retinoic acid receptor antagonists (e.g., 4-[5,6-Dihydro-5,5-dimethyl-8-(quinolin-3- yl)naphthalen-2-carboxamido] benzoic acid (also referred to as BMS195614)), and DNA methyltransferase inhibitors (e.g., 5-azacytidine (5-aza)) (see, e.g., Zhu GJ et al. (2020) Aldh inhibitor restores auditory function in a mouse model of human deafness. PLOS Genetics 16(9): el009040 and Deng X et al., Generation of new hair cells by DNA methyltransferase (Dnmt) inhibitor 5-azacytidine in a chemically-deafened mouse model. Sci Rep. 2019 May 29;9(1):7997, the contents of which are herein incorporated by reference in their entirety).42MF-364562974Docket No.: 336122000640III. Routes of Administration

[0172] Provided herein are methods of preventing hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss in an individual.

[0173] In some aspects, provided herein are methods of preventing hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects, provided herein are methods of treating hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating hearing loss.

[0174] Additionally, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. Also provided herein are methods of treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating a vestibular disorder in an individual.

[0175] In some aspects, provided herein are methods of preventing a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some aspects, provided herein are methods of treating a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein. In some embodiments, the methods provided herein are useful for preventing and treating vestibular disorders.

[0176] In some embodiments, the agent that increases the expression and / or activity of a Pou4f3 protein is formulated as a pharmaceutical composition containing the appropriate carriers and / or excipients. In some embodiments, the agent is solubilized in a carrier, for example, a viscoelastic carrier, that is introduced locally into the inner ear.43MF-364562974Docket No.: 336122000640

[0177] In some embodiments, the composition is administered to an individual, e.g., an individual being treating for hearing loss and / or a vestibular disorder, using a local route of administration. Such local routes of administration include administering the composition into the ear of an individual and / or the inner ear of an individual, for example, by injection and / or using a pump.

[0178] In some embodiments, the composition is injected into the ear (e.g., auricular administration), such as into the luminae of the cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani). For example, the composition is administered by intratympanic injection (e.g., into the middle ear), and / or injections into the outer, middle, and / or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into human ears. Injection can be, for example, through the round window of the ear or through the cochlea capsule. In some embodiments, the composition is administered directly to the inner ear of the individual. In some embodiments, the composition is administered through the posterior semicircular canal of the ear of the individual. In some embodiments, the composition is administered through the round window membrane of the ear of the individual. In some embodiments, the composition is administered to the ear of the individual via a cochleostomy (e.g., via a surgical procedure that involves forming an opening into the cochlea). In such embodiments, the composition can be injected into the cochlea with or without a pump. In some embodiments, the composition is administered to one ear of the individual. In some embodiments, the composition is administered to both ears of the individual.

[0179] In some embodiments, the composition is administered to the inner ear using a catheter or pump. A catheter or pump can, for example, direct the agent into the cochlea luminae or the round window of the ear. Exemplary drug delivery systems suitable for administering one or more compounds into an ear, e.g., a human ear, are described in U.S. Patent Publication No. 2006 / 0030837 and U.S. Pat. No. 7,206,639, the contents of which are herein incorporated by reference in their entirety. In certain embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and / or inner ear) of an individual during a surgical procedure.

[0180] Having generally described the compositions, methods, and processes of this disclosure, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the disclosure and are not intended to limit the scope of the invention as defined by the claims.44MF-364562974Docket No.: 336122000640EXAMPLES

[0181] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.

[0182] Abbreviations: AAV2 (adeno-associated virus type 2); ABR (auditory brainstem response); ARHL (age-related hearing loss); ANOVA (Analysis of Variance); CtBP2 (C- terminal-binding protein 2); CMV (cytomegalovirus); dB (decibel); DPOAE (distortion product otoacoustic emission); hair cell(s) (hair cell(s)); IHC(s) (inner hair cell(s)); MYO7A (Myosin 7A); NIHL (noise-induced hearing loss); OHC(s) (outer hair cell(s)); PSCC (posterior semicircular canal); PTS (permanent threshold shift); and SPL (sound pressure level).Example 1: Pou4f3 for Protection and Treatment of Noise Induced Hearing Loss and Age-Related Hearing LossMethodsPou4f3 expression vector

[0183] The AAV-Pou4f3 vector that was used in this study includes adeno-associated virus type 2 (AAV2); a cytomegalovirus (CMV) promoter; and the mouse Pou4f3 cDNA (AAV2- CMVp-mPou4f3). The sequence of mouse Pou4f3 protein used in this experiment is set forth in SEQ ID NO: 2 (Table 1).Administration ofAAV-Pou4f3 into adult inner ear

[0184] Mice were anaesthetized by intraperitoneal injection of xylazine (10 mg / kg) and ketamine (100 mg / kg). Before surgery, the postauricular region of the mice was gently shaved and disinfected by 10% povidone-iodine. A 10-mm post- auricular incision was made, the pinna and the sternocleidomastoid muscle were extracted to expose the PSCC. The PSCC was perforated by a Bonn micro probe (Fine Science Tools, Foster City, CA). A fine polyimide tube (inner diameter 0.0039 inch, outer diameter 0.0050 inch, MicroLumen, Tampa, FL), which was connected to a glass micropipette and held by a Nanoliter 2000 micromanipulator, was used to deliver AAV. The tip of the tube was inserted into the posterior semicircular canal (PSCC), and the hole was sealed by tissue adhesive. The total volume of virus delivered was 1 pl for each injection at the speed of 100 nl / min. After injection, the tubing was cut and sealed by tissue adhesive. The skin was closed with 7 / 0 suture.45MF-364562974Docket No.: 336122000640Noise exposure

[0185] Mice were exposed to free-field noise while awake and unrestrained, in a small reverberant chamber. An 1-20 kHz octave-band noise was presented for 2 h at 110 dB SPL. The exposure stimulus was generated by a custom white-noise source, filtered (Brickwall Filter with a 60 dB / octave slope), amplified, and delivered through an exponential horn fitted securely to a hole in the top of a reverberant box.Acoustic Testing

[0186] Mice were anaesthetized with xylazine (10 mg / kg) and ketamine (100 mg / kg) intraperitoneally (i.p.). Acoustic stimuli were delivered through a custom acoustic assembly consisting of two miniature dynamic electrostatic earphones to generate primary tones and a miniature microphone to record ear-canal sound pressure near the eardrum. Custom Lab VIEW software controlling National Instruments 24-bit soundcards generated all auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) stimuli and recorded all responses.

[0187] For ABR measurements, needle electrodes were inserted at the vertex and ventral edge of the pinna, with a ground reference near the tail. ABR potentials were evoked with 5-ms tone pips (0.5-ms rise-fall with a cos2 onset, delivered at 35 / s). The response was amplified 10,000- fold, filtered (100 Hz-3 kHz passband), digitized, and averaged (1,024 responses) at each SPL. The sound level was raised in 10 dB steps from 30 dB below threshold up to 100 dB SPL at frequencies from 5.66-32 kHz (in half-octave steps). Following visual inspection of stacked waveforms, “threshold” was defined as the lowest sound pressure level (SPL) at which any wave could be detected. In general, thresholds were defined by three independent observers. Wave 1 amplitude was defined as the difference between the average of the 1-ms pre-stimulus baseline and the wave 1 peak (Pl), after additional high-pass filtering to remove low-frequency baseline shifts.

[0188] For DPOAE measurements, the cubic distortion product was measured in response to primaries fl and f2. The primary tones were set so that the frequency ratio (f2 / fl) was 1.2 and so that the f2 level was 10 dB below the fl level. For each f2 / f 1 primary pair, primaries were swept in 5-dB steps from 20 dB SPL to 80 dB SPL (for f2). At each level, the amplitude of the DPOAE at 2fl-f2 was extracted from the averaged spectra, along with the noise floor. Threshold was computed by interpolation as the f2 level required to produce a DPOAE at 5 dB SPL.46MF-364562974Docket No.: 336122000640Immuno chemistry

[0189] Injected and non-injected cochleae were removed after animals were sacrificed by CO2 inhalation. Temporal bones were fixed in 4% paraformaldehyde at 4 °C overnight, then decalcified in 120 mM EDTA for at least 2 days. The cochleae were dissected in pieces from the decalcified tissue for whole-mount immunofluorescence. Tissues were infiltrated with 0.5% Triton X-100 and blocked with 10% donkey serum for 30 min before applying the primary antibody. Rabbit anti-MYO7A (1:500; #25-6790, Proteus BioSciences); and goat anti- CtBP2 (1:350; BD-Bioscience); and anti-Pou4f3 (1:50; Santa Cruz) were used at room temperature overnight. The second antibody was incubated for 1 h at room temperature after three rinses with Hanks' Balanced Salt Solution (HBSS). Phalloidin-FITC (1:500; Invitrogen), and all Alexafluor secondary antibodies were from Invitrogen at a 1:1000 dilution. Specimens were mounted in ProLong Gold Antifade Mountant medium (P36930, Life Technologies).Confocal microscopy

[0190] Confocal microscopy was performed using a Leica TCS SP8 with Leica Application Suite Advanced Fluorescence (LAS AF) software V2.6.0. Sequential scanning with different laser channels was used for image acquisitions. Confocal images were processed using ImageJ package (www:\\imagej.nih.gov\ij). Z-stacks were acquired by maximum intensity projections of z-stacks for each segment by image! (NIH image), and composite images showing the whole cochlea were constructed in ImageJ. IHCs and OHCs were counted in the cochlear regions that respond to different sound frequencies, and any segments containing dissection-related damage were omitted from further analysis. qRT-PCR

[0191] After noise exposure and subsequent AAV-Pou4f3 injection and hearing testing, the experimental mice were sacrificed. The cochlear tissues including the organ of Corti, spiral limbus, and lateral wall were dissected out from the temporal bones of the adult cochleae in the treated ears (AAV-Pou4f3 injected) and untreated ears (contralateral un-injected). Total RNA was extracted from cochleae using RNeasy Mini Kits from Qiagen (Valencia, CA). cDNA was synthesized using reverse transcriptase (Takara) with random primers. Real-time quantitative PCR amplification reactions were carried out using QuantiTeck SYBR Green PCR kit (Takara) on an ABI StepOnePlus system (Applied Biosystem) with StepOne software V2.3. All reactions were carried out in duplicates with the expression of the gene normalized using Gapdh as the endogenous housekeeping control gene.47MF-364562974Docket No.: 336122000640Statistical Analysis

[0192] The Prism 10 statistical package (GraphPad Software, Inc) was used in data processing. For ABRs, DPOAEs, and Wavel Amplitudes, statistical analyses were performed by two-way ANOVA with Bonferroni corrections for multiple comparisons, (p<0.05 was considered significant).

[0193] To count the number of CtBP2 in the IHCs, the apex (75-100% of the length of cochlear duct from the hook); the apex-mid (50-75% of the length of cochlear duct from the hook) and mid-base (25-50% of the length of cochlear duct from the hook) were included. Phalloidin+ stereocilia bundles and MYO7A+ HCs were counted per 100 pm for each treated group. Statistical analyses were performed by two-way ANOVA with Bonferroni corrections for multiple comparisons, (p<0.05 was considered significant). In epigenetic modulators plus AAV-Pou4f3 treated samples, controls were 0.9% sterile saline-injected cochleae; in AAV- Pou4f3 alone treated samples, controls were the un-treated contralateral cochleae.

[0194] Data were presented as mean ± SEM. Student’ s t- test was used to compare two groups (p<0.05 was considered significant). ANOVA analysis with Bonferroni corrections for multiple comparisons test was used to compare three or more groups (p<0.05 was considered significant).Example 2: Results

[0195] The following Example demonstrates that Pou4f3 overexpression in the inner ear hair cells restores hearing loss caused by noise exposure and prevents age-related progressive hearing loss.Pou4f3 expression recovers hearing from severe noise induced hearing loss

[0196] In this study, overexpression of Pou4f3 was assessed for its ability to restore hearing following severe noise induced hearing loss. The methods described in Example 1 were used. Briefly, mice were exposed to free-field noise while awake and unrestrained in a small reverberant chamber. Eight days following noise exposure, AAV-Pou4f3 was administered to the inner ear of each mouse. The contralateral ear of each mouse served as a control. Hearing was assessed 1 month and 4 months after AAV-Pou4f3 treatment (FIG. 1).

[0197] Wave 1 amplitude represents the summed activity of the auditory nerve fibers contacting inner hair cells (IHCs) and can be used to assess the individual for potential cochlear synaptopathy. As shown in FIG. 2 and FIG. 3, the injected ears showed a significant increase in Wave 1 amplitude at 16, 22.6, and 32 kHz at lOOdB SPL compared to the untreated48MF-364562974Docket No.: 336122000640 contralateral ears, thus demonstrating that Pou4f3 overexpression can recover the synaptic connections between the auditory nerve fibers and the IHCs one month following AAV-Pou4f3 treatment.

[0198] Auditory brainstem response (ABR) threshold, which provides a metric for overt hearing loss, was also tested. As shown in FIG. 4A, the injected ears showed a significant decrease in ABR threshold at 8 and 16 kHz compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression can recover hearing one month following AAV- Pou4f3 treatment.

[0199] The distortion product otoacoustic emission (DPOAE) read-out depends on the biological motors in outer hair cells (OHCs) which amplify sound-evoked cochlear vibration. DPOAE threshold can thus provide a measure of OHC damage. As shown in FIG. 4B, the injected ears showed a non- significant decrease in DPOAE threshold at several tested frequencies (e.g., 11.32, 16, 22.6, and 32 kHz) compared to the untreated contralateral ears, thus suggesting that Pou4f3 overexpression may recover damaged OHCs one month following AAV-Pou4f3 treatment.

[0200] Wave 1 amplitudes, ABR thresholds, and DPOAE thresholds of the mice were also tested 4 months after AAV-Pou4f3 administration to assess the long-term therapeutic effects. As shown in FIG. 5 and FIG. 6, the injected ears showed a significant increase in Wave 1 amplitude at 11.32 kHz compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression from AAV-Pou4f3 treatment can provide long term recovery of the synaptic connections between the auditory nerve fibers and the IHCs.

[0201] As shown in FIG. 7A, the injected ears showed a non-significant decrease in ABR threshold at several tested frequencies (e.g., 8, 11.32, 16, 22.6, and 32 kHz) compared to the untreated contralateral ears, thus suggesting that Pou4f3 overexpression can provide long term recovery of hearing. As shown in FIG. 7B, the injected ears did not demonstrate a significant decrease in DPOAE threshold at the tested frequencies after 4 months.

[0202] Overall, the results from this study demonstrate that Pou4f3 overexpression can recover hearing from severe noise induced hearing loss and this effect can be observed long term.Pou4f3 expression before noise damage protects hearing from severe noise induced hearing loss

[0203] In this study, overexpression of Pou4f3 was assessed for its ability to serve as a prophylactic against severe noise induced hearing loss. The methods described in Example 149MF-364562974Docket No.: 336122000640 were used. Briefly, AAV-Pou4f3 was administered to the inner ear of each mouse. The contralateral ear of each mouse served as a control. Eight days following AAV-Pou4f3 administration, mice were exposed to free-field noise while awake and unrestrained in a small reverberant chamber. Hearing was assessed 11.5 weeks after noise exposure (FIG. 8).

[0204] Wave I amplitudes of the mice were measured. As shown in FIG. 9 and FIG. 10, the injected ears showed a significant increase in Wave 1 amplitude at 16 and 22.6 kHz at lOOdB SPE compared to the untreated contralateral ears, thus showing that AAV-Pou4f3 treatment alone before noise exposure greatly preserves Wavel Amplitudes against HOdB permanent threshold shift (PTS) noise damage. Overall, FIG. 9 and FIG. 10 demonstrate that Pou4f3 overexpression can provide prophylactic protection of the synaptic connections between the auditory nerve fibers and the IHCs.

[0205] ABR threshold was also measured. As shown in FIG. 11 A, the injected ears showed a significant decrease in ABR threshold at several frequencies (z.e., 11.32, 16, 22.6, and 32 kHz) compared to the untreated contralateral ears, thus demonstrating that AAV-Pou4f3 treatment alone before noise exposure greatly preserves ABR against HOdB PTS noise damage. Overall, FIG. 11A shows that Pou4f3 overexpression can provide prophylactic protection of hearing.

[0206] Finally, DPOAE thresholds of the mice were measured. As shown in FIG. 1 IB, the injected ears showed a significant decrease in DPOAE threshold at 16 and 22.6 kHz compared to the untreated contralateral ears, thus demonstrating that AAV-Pou4f3 treatment alone before noise exposure greatly preserves DPOAE against HOdB permanent PTS noise damage. Overall, FIG. 11B shows that Pou4f3 overexpression can provide prophylactic protection of OHCs.

[0207] Overall, the results from this study demonstrate that Pou4f3 overexpression prior to noise exposure can protect hearing from severe noise induced hearing loss.Pou4f3 expression recovers hearing from age-related hearing loss

[0208] In this study, overexpression of Pou4f3 was assessed for its ability to recover hearing caused by age-related hearing loss. The methods described in Example 1 were used. Briefly, AAV-Pou4f3 was administered to the inner ear of C57Bl / 6j mice. The contralateral ear of each mouse served as a control. C57Bl / 6j mice have a genetic mutation in the Cdh23 gene which encodes Cadherin 23, a glycoprotein necessary for both development and maintenance of the hair bundle and tip links of the hair cells and is therefore essential for mechanotransduction. Mice homozygous for this mutation show a progressive high frequency hearing loss starting at50MF-364562974Docket No.: 336122000640 about 3 months of age that eventually results in severe hearing loss at one year of age (10-12). Hearing was assessed 2 and 4 months after AAV-Pou4f3 treatment (FIG. 12).

[0209] IHC synapse count declines steadily throughout life and is associated with age-related hearing loss. Accordingly, Wave I amplitudes of the mice were measured to assess cochlear synaptopathy. As shown in FIG. 13 and FIG. 14, the injected ears showed a significant increase in Wave 1 amplitude at several frequencies (z.e., 5.66, 8, 11.32, 16, and 22.6 kHz) at lOOdB SPL compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression can recover the synaptic connections between the auditory nerve fibers and the IHCs that are lost during aging.

[0210] ABR threshold was also measured. As shown in FIG. 15 A, the injected ears showed a significant decrease in ABR threshold at 22.6 and 32 kHz compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression can recover hearing from age-related hearing loss.

[0211] Finally, DPOAE thresholds of the mice were measured. As shown in FIG. 15B, the injected ears showed a non-significant decrease in DPOAE threshold at 11.32, 16, 22.6, and 32 kHz compared to the untreated contralateral ears, thus suggesting that Pou4f3 overexpression can recover OHCs that are damaged during aging.

[0212] Wave 1 amplitudes, ABR thresholds, and DPOAE thresholds of the mice were also measured 4 months after AAV-Pou4f3 administration to assess the long-term therapeutic effects. As shown in FIG. 16 and FIG. 17, the injected ears showed a significant increase in Wave 1 amplitude at 5.66, 8, 11.32, and 22.6 kHz at lOOdB SPL compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression can provide long term recovery of the synaptic connections between the auditory nerve fibers and the IHCs that are lost during aging.

[0213] As shown in FIG. 18 A, the injected ears showed a significant decrease in ABR threshold at 22.6 kHz compared to the untreated contralateral ears, thus demonstrating that Pou4f3 overexpression can provide long term recovery of hearing from age-related hearing loss. As shown in FIG. 18B, the injected ears showed a significant decrease in DPOAE threshold at 22.6 kHz compared to the untreated contralateral ears, thus demonstrating Pou4f3 overexpression can provide long term recovery of OHCs that are damaged during aging.

[0214] Overall, the results from this study demonstrate that Pou4f3 overexpression can recover hearing from age-related hearing loss and that this effect can be observed long term.51MF-364562974Docket No.: 336122000640Hair Cell Mark Analysis

[0215] In this study, overexpression of Pou4f3 was assessed for its ability to restore cochlear morphology. The methods described in Example 1 were used. Briefly, mice were exposed to free-field noise while awake and unrestrained in a small reverberant chamber. Three to eight days after noise exposure, AAV-Pou4f3 was administered to the inner ear of each mouse. The contralateral ear of each mouse served as a control. Two to four months after AAV-Pou4f3 treatment, mice were sacrificed, and the cochleae of each ear was removed and processed for analysis. Injected and non-injected cochleae samples were analyzed for Pou4f3, Myosin 7A (MYO7A), C-terminal-binding protein 2 (CtBP2), and F-Actin (FIG. 19). Hair cell bundle genes were also analyzed by qRT-PCR.

[0216] Treated and untreated ear samples were stained for Pou4f3 two months after AAV- Pou4f3 administration to assess whether the treatment induces overexpression of the Pou4f3 protein. As shown in FIGs. 20A-20B, the injected ear sample showed a significant increase in Pou4f3 overexpression in cochlear hair cells compared to the cochlear hair cells of the untreated contralateral ear sample. In particular, the AAV-Pou4f3 expression level was about 2-fold more than that of endogenous Pou4f3 (FIG. 20B). Overall, FIGs. 20A-20B demonstrate that AAV-Pou4f3 administration to the inner ear results in Pou4f3 overexpression in cochlear hair cells.

[0217] Treated and untreated ear samples were also stained for the hair cell marker, MYO7A, in the apex, middle (mid), and base of the cochlea. As shown in FIG. 21 A, the injected ear sample showed increased MYO7A in the cochlea compared to the untreated contralateral ear sample, particularly in the base of the cochlea. The arrows in FIG. 21 A (untreated; top panel) show the areas where MYO7A is missing due to the loss of hair cells following noise damage. As shown in FIGs. 2 IB and 21C, the number of OHCs and IHCs were preserved in each region of the cochlea following AAV-Pou4f3 treatment. A significant decrease in the number of OHCs was observed in the base of the cochlea for the untreated contralateral ear samples compared to the injected ear samples (FIG. 2 IB). Overall, FIGs. 21A-21C demonstrate that AAV-Pou4f3 administration preserves hair cells from noise damage.

[0218] Treated and untreated ear samples were also stained for the presynaptic marker, CtBP2, in the apex, middle (mid), and base of the cochlea (FIG. 22A). As shown in FIG. 22A, morphological analysis of the pre- synaptic ribbon (CtBP2 labeling) confirmed a robust preservation of synapses in the IHCs after noise exposure 3 months later. As shown in FIG. 22B, a significant decrease in CtBP2 per IHC was observed in each region of the cochlea for52MF-364562974Docket No.: 336122000640 the untreated contralateral ear samples compared to the injected ear samples. Overall, FIGs. 22A-22B demonstrate that AAV-Pou4f3 administration preserves the hair cell synaptic ribbons from noise damage.

[0219] Treated and untreated ear samples were also stained for the hair cell stereocilia marker, F-Actin (Phalloidin), in the apex, middle (mid), and base of the cochlea. As shown in FIG. 23A, the injected ear sample showed increased F-Actin in the cochlea compared to the untreated contralateral ear sample. The arrows in FIG. 23A (untreated; right panel) show the areas where F-Actin is missing due to the loss of stereocilia bundles following noise damage. As shown in FIG. 23B, the percentage of IHCs without stereocilia bundles increased significantly in the middle region of the cochlea of the untreated contralateral ear samples compared to the injected ear samples. Overall, FIGs. 23A-23B demonstrate that AAV-Pou4f3 administration preserves hair cell stereocilia bundles from noise damage.

[0220] Finally, hair cell bundle genes were analyzed by qRT-PCR for the injected ear sample and the untreated contralateral ear sample. As shown in FIG. 24, Pou4f3 overexpression led to increased expression of multiple inner ear hair cell bundle genes in vivo. In particular, inner ear bundle genes Atp8bl, Myo7a, Espn, Cdh23, Pcdhl5, Tmcl, Ptprq, Strc, Ckb, Myol5, Tmie, Atp2b2, and Ush2a were up-regulated.

[0221] Overall, the results from this study demonstrate that AAV-Pou4f3 administration to the inner ear results in Pou4f3 overexpression in cochlear hair cells and that this overexpression preserves hair cells, hair cell synaptic ribbons, and stereocilia bundles from noise damage.Atohl expression does not restore hearing from severe noise induced hearing loss

[0222] In this study, overexpression of another transcription factor, Atohl, was assessed for its ability to restore hearing following severe noise induced hearing loss. Atohl, a master transcription factor, is required for the differentiation of the inner ear hair cells during normal development and for hair cell regeneration in newborn and adult mice. The methods described in Example 1 were used. Briefly, mice were exposed to free-field noise while awake and unrestrained in a small reverberant chamber. Ten days following noise exposure, AAV-Atohl was administered to the inner ear of each mouse. The contralateral ear of each mouse served as a control. Hearing was assessed 3 weeks after AAV-Atohl treatment (FIG. 25).

[0223] Wave 1 amplitudes of the mice were measured. As shown in FIG. 26 and FIG. 27, the injected ears did not show a significant increase in Wave 1 amplitude at any frequency compared to the untreated contralateral ears, thus demonstrating that Atohl overexpression53MF-364562974Docket No.: 336122000640 does not recover the synaptic connections between the auditory nerve fibers and the IHCs following noise damage.

[0224] ABR thresholds of the mice were also measured. As shown in FIG. 28A, the injected ears did not show a significant decrease in ABR threshold at any frequency compared to the untreated contralateral ears, thus demonstrating that Atohl overexpression does not recover hearing from noise damage.

[0225] Finally, DPOAE thresholds of the mice were measured. As shown in FIG. 28B, the injected ears did not show a significant decrease in DPOAE threshold at any frequency compared to the untreated contralateral ears. In fact, a significant decrease in the DPOAE threshold was observed at 16, 22.6, and 32 kHz for the untreated contralateral ears compared to the injected ears. Accordingly, FIG. 28B demonstrates that Atohl overexpression does not recover OHCs from noise damage.

[0226] Overall, the results from this study demonstrate that, unlike Pou4f3 overexpression, Atohl overexpression alone cannot recover hearing from severe noise induced hearing loss.Conclusion

[0227] At present, there is no FDA-approved drug with demonstrated efficacy to treat noise- induced hearing loss (NIHL) and age-related hearing loss (ARHL). Pou4f3 has previously demonstrated its critical role in the survival of inner ear hair cells during embryonic development (1-3). Studies have shown that Pou4f3 regulates inner ear hair cell survival via its target gene Gfil (4). The co-regulation of the key inner ear transcription factors Pou4f3 / Atohl / Gfil in the embryonic cell line and transgenic mouse epithelium can mimic the development stage for immature inner ear hair cell-like cell regeneration (5-7). Knockout of the Pou4f3 leads to hair cell death and is detrimental to hearing (8). Yet, none of the publication above can recover / treat hearing loss (9). Demonstrated herein, for the first time, the overexpression of the key transcription factor, Pou4f3, in the inner ear cochlear hair cell is efficacious to attenuate noise induced hearing loss (NIHL), repair synaptopathy, and ameliorate progressive hearing decline in age-related hearing loss (ARHL). In contrast, similar experiments testing a separate transcription factor, Atohl, did not show any treatment effect on NIHL and ARHL.References1) Singh, S. et al. Highly variable hearing loss due to POU4F3 (c.37del) is revealed by longitudinal, frequency specific analyses. Eur J Hum Genet 31, 815-823 (2023).54MF-364562974Docket No.: 3361220006402) Yu, H.V. et al. POU4F3 pioneer activity enables ATOH1 to drive diverse mechanoreceptor differentiation through a feed-forward epigenetic mechanism. Proc Natl Acad Sci U S A 118 (2021).3) Scheffer, D.I., Shen, J., Corey, D.P. & Chen, Z.Y. Gene Expression by Mouse Inner Ear Hair Cells during Development. JNeurosci 35, 6366-6380 (2015).4) Hertzano, R. et al. Transcription profiling of inner ears from Pou4f3(ddl / ddl) identifies Gfil as a target of the Pou4f3 deafness gene. Hum Mol Genet 13, 2143-2153 (2004).5) Iyer, A.A. et al. Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals. Elife 11 (2022).6) McGovern, M.M. et al. Expression of Atohl, Gfil, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells. Proc Natl Acad Sci U S A 121, e2304680121 (2024).7) Costa, A. et al. Generation of sensory hair cells by genetic programming with a combination of transcription factors. Development 142, 1948-1959 (2015).8) Singh, J., Randle, M.R., Walters, B.J. & Cox, B.C. The transcription factor Pou4f3 is essential for the survival of postnatal and adult mouse cochlear hair cells and normal hearing. Front Cell Neurosci 18, 1369282 (2024).9) Zhang, L. et al. AAV-mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration. Adv Sci (Weinh) 11, e2304551 (2024).10) Kendall, A. & Schacht, J. Disparities in auditory physiology and pathology between C57BL / 6J and C57BL / 6N substrains. Hear Res 318, 18-22 (2014).11) Burghard, A.L., Morel, N.P. & Oliver, D.L. Mice heterozygous for the Cdh23 / Ahll mutation show age-related deficits in auditory temporal processing. Neurobiol Aging 81, 47- 57 (2019)12) Someya, S. et al. Sirt3 mediates reduction of oxidative damage and prevention of age- related hearing loss under caloric restriction. Cell 143, 802-812 (2010).55MF-364562974

Claims

Docket No.: 336122000640CLAIMSWhat is claimed is:

1. A method of preventing and / or treating hearing loss in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

2. A method of preventing and / or treating a vestibular disorder in an individual comprising administering to the individual a composition comprising an agent that increases the expression and / or activity of a Pou4f3 protein.

3. The method of claim 1 or 2, wherein the agent is a Pou4f3 protein.

4. The method of claim 1 or 2, wherein the agent is a nucleic acid encoding a Pou4f3 protein.

5. The method of claim 4, wherein the nucleic acid encoding the Pou4f3 protein is an RNA molecule.

6. The method of claim 4, wherein the nucleic acid encoding the Pou4f3 protein is a DNA molecule.

7. The method of claim 1 or 2, wherein the agent activates a Pou4f3 gene.

8. The method of claim 7, wherein the agent is a small activating RNA.

9. The method of claim 7, wherein the agent is a small molecule.

10. A method of preventing and / or treating hearing loss in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein.

11. A method of preventing and / or treating a vestibular disorder in an individual comprising administering to the ear of the individual a composition comprising a Pou4f3 protein or a nucleic acid sequence encoding a Pou4f3 protein.56MF-364562974Docket No.: 33612200064012. The method of claim 10 or 11, wherein the composition comprises a viral vector comprising the nucleic acid sequence encoding the Pou4f3 protein.

13. The method of any one of claims 1-12, wherein the composition is administered to the ear of the individual.

14. The method of any one of claims 1-13, wherein the composition is administered to a cochlear cell in the ear of the individual.

15. The method of claim 14, wherein the cochlear cell is selected from the group consisting of a stria vascularis cell, a hair cell, a supporting cell, and a spiral ganglion neuron.

16. The method of claim 15, wherein the cochlear cell is a hair cell.

17. The method of claim 16, wherein the hair cell is an inner hair cell.

18. The method of claim 16, wherein the hair cell is an outer hair cell.

19. The method of any one of claims 1, 2, and 13-18, wherein the agent is a viral vector comprising a nucleic acid sequence encoding a Pou4f3 protein.

20. The method of any one of claims 10-19, wherein the nucleic acid sequence encoding the Pou4f3 protein is operably linked to a promoter sequence.

21. The method of claim 20, wherein the promoter sequence is selected from the group consisting of a constitutive promoter sequence, an inducible promoter sequence, and a tissue- specific promoter sequence.

22. The method of claim 21, wherein the promoter sequence is a constitutive promoter sequence.

23. The method of claim 22, wherein the constitutive promoter sequence is selected from the group consisting of a human cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, a simian virus 40 (SV40) promoter sequence, and a mammalian elongation factor la (EFla) promoter sequence.

24. The method of claim 23, wherein the constitutive promoter sequence is a CMV promoter sequence.57MF-364562974Docket No.: 33612200064025. The method of claim 21, wherein the promoter sequence is a tissue-specific promoter sequence.

26. The method of claim 25, wherein the tissue-specific promoter sequence is selected from the group consisting of a stria vascularis cell- specific promoter sequence, a hair cellspecific promoter sequence, a supporting cell-specific promoter sequence, and a spiral ganglion neuron-specific promoter sequence.

27. The method of any one of claims 12-26, wherein the viral vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vesicular stomatitis virus (VSV) vector, a herpes simplex virus (HSV) vector, a vaccinia virus vector, a pox virus vector, an influenza virus vector, a respiratory syncytial virus vector, a parainfluenza virus vector, a foamy virus vector, and a retrovirus vector.

28. The method of claim 27, wherein the viral vector is an adeno-associated virus type 2 (AAV2) vector.

29. The method of any one of claims 10-28, wherein the Pou4f3 protein encoded by the nucleic acid sequence comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

30. The method of claim 29, wherein the Pou4f3 protein encoded by the nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 4.

31. The method of any one of claims 1-30, wherein the composition is administered directly to the inner ear of the individual.

32. The method of any one of claims 1-30, wherein the composition is administered through the posterior semicircular canal of the ear of the individual.

33. The method of any one of claims 1-30, wherein the composition is administered through the round window membrane of the ear of the individual.

34. The method of any one of claims 1-33, wherein the composition is administered to both ears of the individual.58MF-364562974Docket No.: 33612200064035. The method of any one of claims 1-34, further comprising determining the auditory brainstem response (ABR) threshold of the individual at a frequency before and after the individual has received the composition.

36. The method of claim 35, wherein the method results in a decrease in the ABR threshold of the individual at a frequency as compared to the ABR threshold of the individual at the same frequency prior to administration of the composition.

37. The method of any one of claims 1-36, further comprising determining the Wave I amplitude of the individual at a frequency before and after the individual has received the composition.

38. The method of claim 37, wherein the method results in an increase in the Wave I amplitude of the individual at a frequency as compared to the Wave I amplitude of the individual at the same frequency prior to administration of the composition.

39. The method of any one of claims 1-38, further comprising determining the distortion product otoacoustic emission (DPOAE) threshold of the individual at a frequency before and after the individual has received the composition.

40. The method of claim 39, wherein the method results in a decrease in the DPOAE threshold of the individual at a frequency as compared to the DPOAE threshold of the individual at the same frequency prior to administration of the composition.

41. The method of any one of claims 1-40, wherein the method reduces and / or prevents the loss of outer hair cells and / or inner hair cells in the ear of the individual.

42. The method of any one of claims 1-41, wherein the method reduces and / or prevents the loss of synapses between inner hair cells and spiral ganglion neurons in the ear of the individual.

43. The method of any one of claims 1-42, wherein the method reduces and / or prevents the loss of stereocilia in the ear of the individual.

44. The method of any one of claims 1, 3-10, and 12-43, wherein the hearing loss is noise-induced hearing loss.59MF-364562974Docket No.: 33612200064045. The method of any one of claims 1, 3-10, and 12-43, wherein the hearing loss is age-related hearing loss.

46. The method of any one of claims 1, 3-10, and 12-43, wherein the hearing loss is progressive non-syndromic hearing loss (NSHL).

47. The method of claim 46, wherein the individual has a mutation in a POU4F3 gene.

48. The method of any one of claims 1, 3-10, and 12-43, wherein the hearing loss is progressive hearing loss.

49. The method of claim 48, wherein the individual has a mutation in a hair cell bundle gene.

50. The method of claim 48, wherein the individual has a mutation in a CDH23 gene.

51. The method of any one of claims 1, 3-10, and 12-43, wherein the hearing loss is sudden hearing loss.

52. The method of any one of claims 2-9 and 11-43, wherein the vestibular disorder is associated with hearing loss in the individual.

53. The method of any one of claims 2-9 and 11-43, wherein the vestibular disorder is associated with progressive hearing loss.

54. The method of any one of claims 2-9 and 11-43, wherein the vestibular disorder is associated with age-related hearing loss.

55. The method of any one of claims 1-54, wherein the individual exhibits cochlear synaptopathy.

56. The method of any one of claims 1-55, wherein the individual exhibits tinnitus.

57. The method of any one of claims 1-56, wherein the individual is human.60MF-364562974

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