Methods and compositions for regenerating hair cells in the inner ear of adult mammals

Abstract

Provided herein is a method for regenerating hair cells in the inner ear of adult mammals, using a novel combination of agents selected from the group consisting of histone deacetylase (HDAC) inhibitors, one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7 or combinations thereof, Wnt pathway activators and cAMP activators.The methods and compositions can be used to treat subjects with hearing loss or vestibular dysfunction.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 144,883, filed February 2, 2021, the entire contents of which are incorporated by reference herein.

[0002] Federally Sponsored Research or Development This invention was made with Government support under Grant Nos. R01DC006908 and UH3TR002636 awarded by the National Institutes of Health and Grant No. W81XWH1810331 awarded by the Department of Defense. The Government has certain rights in the invention.

[0003] The subject matter disclosed herein relates generally to methods for regenerating hair cells in the inner ear of adult mammals, and more specifically to the use of a unique combination of agents that is amenable to use in clinical practice. [Background technology]

[0004] Worldwide, hearing loss affects 1 in 500 newborns and half of the population over 70 years of age. Despite being the most common dysfunction of the human senses, there is currently no pharmacological treatment for hearing loss. Cochlear hair cells (HCs) in adult mammals, which are responsible for converting sound signals into electrical impulses, have completely lost the ability to spontaneously regenerate after injury. The loss of hair cells in the adult cochlea is thought to be the primary cause of hearing loss. Summary of the Invention

[0005] The present disclosure is based, at least in part, on the discovery of novel combinations of small molecules and inhibitory nucleic acids that induce hair cell regeneration in transgenic mouse models of hearing loss. It has also been demonstrated that the novel combinations described herein can be used to regenerate hair cells in adult wild-type mice with chemically induced hair cell damage.

[0006] Accordingly, an embodiment of the present disclosure provides a method for reprogramming the inner ear of an adult mammal for hair cell regeneration, comprising contacting the inner ear of an adult mammal with an effective amount of a histone deacetylase (HDAC) inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or a combination thereof, under conditions and for a time sufficient to produce a population of progenitor cells in the inner ear of the adult mammal.

[0007] In some embodiments, the HDAC inhibitor is sodium butyrate, trichostatin A, hydroxamic acid, cyclic tetrapeptide, trapoxin B, depsipeptide, benzamide, electrophilic ketone, fatty acid compound, pyroxamide, phenylbutyrate, valproic acid, hydroxamic acid, romidepsin, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589), ene. Selected from the group consisting of tinostat (MS275), CI-994 (N-acetyldinaline, also known as tacedinaline), entinostat (SNDX-275; formerly MS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, gibinostat (ITF2357) and mocetinostat (MGCD0103).

[0008] In some embodiments, the HDAC inhibitor is valproic acid, Trichostatin A, vorinostate (SAHA) or belinostat (PXD101).

[0009] In some embodiments, the one or more inhibitory nucleic acids are small interfering RNA (siRNA), small hairpin RNA (shRNA) or antisense oligonucleotides. In some embodiments, the one or more inhibitory nucleic acids include inhibitory nucleic acids that target Fir and Mxi1.

[0010] In some embodiments, the methods described herein further comprise contacting the mammalian cochlea with a Wnt agonist and / or a cAMP agonist. In some embodiments, the Wnt activator is lithium chloride (LiCl) and / or the cAMP activator is forskolin.

[0011] In some embodiments, the progenitor cells of the population express Six1, Eya1, Gata3, Sox2, Notch1, Hes5, or a combination thereof.

[0012] In some embodiments, the contacting occurs in the subject's inner ear.

[0013] An embodiment of the present disclosure provides a method for treating hearing loss or vestibular dysfunction in a subject, the method comprising administering to the inner ear of a subject in need thereof an effective amount of a histone deacetylase (HDAC) inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7 or a combination thereof; and administering to the inner ear of the subject an effective amount of an Atoh1 activator.

[0014] In some embodiments, the HDAC inhibitor is sodium butyrate, trichostatin A, hydroxamic acid, cyclic tetrapeptide, trapoxin B, depsipeptide, benzamide, electrophilic ketone, fatty acid compound, pyroxamide, phenylbutyrate, valproic acid, hydroxamic acid, romidepsin, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (L BH589), entinostat (MS275), CI-994 (N-acetylglutamic acid, also known as tacedinaline), entinostat (SNDX-275; formerly MS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, gibinostat (ITF2357) and mocetinostat (MGCD0103).

[0015] In some embodiments, the HDAC inhibitor is valproic acid, trichostatin A, vorinostat (SAHA) or belinostat (PXD101).

[0016] In some embodiments, the one or more inhibitory nucleic acids are small interfering RNA (siRNA), small hairpin RNA (shRNA) or antisense oligonucleotides. In some embodiments, the one or more inhibitory nucleic acids target Fir and Mxi1.

[0017] In some embodiments, the Atoh1 activator is a nucleic acid encoding Atoh1. In some embodiments, the nucleic acid encoding Atoh1 is contained in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, herpes simplex viral vector and vaccinia viral vector.

[0018] In some embodiments, the methods described herein further comprise administering to the subject a Wnt agonist and / or a cAMP agonist. In some embodiments, the Wnt activator is lithium chloride (LiCl) and / or the cAMP activator is forskolin.

[0019] In some embodiments, the subject is a human patient with noise-induced permanent hearing loss, drug-induced hearing loss, age-related hearing loss, sudden sensorineural hearing loss, hearing loss due to a viral infection, tinnitus, vestibular dysfunction, or a combination thereof. In some embodiments, the subject is a human.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used.Materials, methods and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief description of the drawings]

[0022] [Figure 1-1] Figure 1 shows data demonstrating that VPA / siFM can reprogram sensory cells in the adult mouse cochlea.Figure 1A: Schematic illustrating the experimental procedure using VPA / siFM in WT mice followed by ad-Atoh1-mcherry in vitro. [Figure 1-2] Data showing that VPA / siFM can reprogram sensory cells in the adult mouse cochlea (Figure 1B): VPA / siFM or vehicle (DMSO)-treated, and ad-Atoh1-mcherry-treated adult (P30) WT mouse cochlea samples were labeled with MYO7A / mcherry. [Figure 1-3] Data showing that VPA / siFM can reprogram sensory cells in the adult mouse cochlea. FIG. 1C: Quantification and comparison showed a significant increase in regenerated HCs in cultured cochleae between VsiFsiM / ad-Atoh1 or vehicle / ad-Atoh1 treated groups. *p<0.05, Student's t-test. Error bars, mean±SEM; n=5 for each group. SE: sensory region; Lib: peripheral region. V: VPA; siF: siFIR; siM: siMxi1. N=5 in each group. Scale bar: 10 μm. [Figure 2-1]FIG. 2A: Schematic illustrating various combinations of small molecules and siRNAs that failed to reprogram and regenerate hair cells in cultured adult mouse cochleae. [Figure 2-2] FIG. 2B: Schematic illustrating various combinations of small molecules and siRNAs that failed to reprogram and regenerate hair cells in cultured adult mouse cochleae. [Figure 2-3] FIG. 2C: Schematic illustrating various combinations of small molecules and siRNAs that failed to reprogram and regenerate hair cells in cultured adult mouse cochleae. [Figure 3-1] Data showing that cocktail (VPA / siFIR / siMxi1) treatment regenerates HCs in cultured adult mouse utricles following neomycin injury. Figure 3A: Schematic illustrating the experimental procedure for utricle samples treated with neomycin. Figure 3B: Neomycin-treated adult wild-type mouse utricle samples showed a reduction in hair cells (stained with MYO7A) compared to untreated utricle samples. [Figure 3-2] Data showing that cocktail (VPA / siFIR / siMxi1) treatment regenerates HCs in cultured adult mouse utricles following neomycin injury. Figure 3C: Schematic illustrating the experimental procedure for utricle samples treated with neomycin and cocktail (VPA / siFIR / siMxi1). Figure 3D: After 5 days of neomycin treatment, cultured utricles subsequently treated with VPA / siFIR / siMxi1 showed an increase in hair cells (MYO7A positive) over vehicle (DMSO)-treated utricles. SOX2 labels both hair cells and supporting cells. [Figure 4-1] Figure 4 shows data demonstrating the reprogramming effect of the cocktail in vitro.Figure 4A: Schematic illustrating the experimental procedure of cochlear samples treated with a combination of small molecules (VLFsiFsiM). [Figure 4-2]Figure 4B: Data showing the reprogramming effect of the cocktail in vitro. Figure 4B: qRT-PCR in cultured adult WT mouse cochleae after 4 days of cocktail (VLFsiFsiM) treatment. A select group of inner ear progenitor genes were upregulated, including Six1, Eya1, Gata3 and Sox2, Notch1 and its target Hes5. Some inner ear progenitor genes (Hes1, Foxg1 and Dlx5) showed little change in expression level. No stem cell genes (Fut4, Nanog and Alpl) or differentiation genes (Prox1, P27kip1 or Cdkn1b) were upregulated. *p<0.05, **p<0.01, ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM, n=3. n is the number of biologically independent samples. V:VPA;L:LiCl;F:FSK;siF:siFIR;siM:siMxi1. [Figure 5-1] Figure 5 shows data showing that the small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Figure 5A: Schematic illustrating the experimental procedure of cochlear samples treated with the application of the small molecule and siRNA combination (VLFsiFsiM) in Atoh1-GFP mice in vitro and HC induction with ad-Atoh1. [Figure 5-2] Data showing that the small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Figures 5B-5C: VLFsiFsiM (or vehicle DMSO) / ad-Atoh1-treated adult (P30) Atoh1-GFP mouse cochlear samples were labeled with MYO7A (dark grey) / Atoh1-GFP (light grey) in the sensory epithelium region. [Figure 5-3]Data showing that small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Figure 5D-5G: Quantification and comparison showed an increase in regenerated HCs (GFP+MYO7A labeled) in the sensory epithelium (SE) or peripheral region (Lib) of cultured cochleae in DLFsiFsiM / ad-Atoh1 treated samples compared to vehicle / ad-Atoh1 treated samples. The number of infected cells (GFP positive) was similar between the two groups. T, Tamo: 4-hydroxytamoxifen; V: VPA; L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. **p<0.01, ****p<0.0001, Student's t-test. Error bars, mean ± SEM; N=5-6 in each group. Scale bar: 10 μm. [Figure 5-4] Data showing that the small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Figures 5H-5I: VLFsiFsiM / ad-Atoh1-treated adult (P30) Atoh1-GFP mouse cochlea samples were labeled with Atoh1-GFP / FM1-43 / MYO7A, an indicator of functional hair cells. [Figure 5-5] Data showing that the small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Figure 5J: Schematic illustrating the experimental procedure of lineage tracing by Tamo induction of Sox2CreER / tdT and HC induction by ad-Atoh1 with VLFsiFsiM in vitro. [Figure 5-6] Data showing that small molecule and siRNA cocktail (VLFsiFsiM) induced robust hair cell regeneration. Fig. 5K: Tamo / VLFsiFsiM / ad-Atoh1 treated adult (P30) Sox2CreER / tdT mouse cochlear samples were labeled with MYO7A / Sox2-tdT. Fig. 5L: Enlarged view from (Fig. 5K). Arrows indicate MYO7A / Sox2-tdT double positive cells. Arrowheads indicate MYO7A+ / Sox2-tdT cells, demonstrating that regenerated hair cells were derived from supporting cell transdifferentiation. [Figure 6]1 shows data demonstrating that various HDAC inhibitors can be used in combination with Myc modulator siRNA to efficiently regenerate hair cells in the adult cochlea in vitro after Ad-Atoh1 infection. L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. [Figure 7-1] Figures 7A, 7B: Data showing that siRNA against the Myc modulator Fbxw7, alone or in combination with siRNA against the Myc modulators Fir and Mxi1 and the HDAC inhibitor VPA, promotes hair cell regeneration with different efficiency in the adult wild-type cochlea in vitro. [Figure 7-2] FIG. 7C: Data showing that siRNA against the Myc modulator Fbxw7, alone or in combination with siRNA against the Myc modulators Fir and Mxi1 and the HDAC inhibitor VPA, promotes hair cell regeneration with different efficiency in the adult wild-type cochlea in vitro. [Figure 8-1] Figure 8 shows data from an in vivo severe HC loss model. Figure 8A: Schematic illustrating the experimental procedure of C57BL / 6j mice treated with Kanamycin / Furosemide in vivo. Figure 8B: Kana / Furo treated adult wild type mouse cochlear samples stained with MYO7A / SOX2. Scale bar: 10 μm. [Figure 8-2] Figure 8C: Quantification and comparison of hair cells and supporting cells between freshly amputated and Kana / Furo treated ears (HC / SC). Data showed that after Kanamycin / Furo treatment, most outer hair cells were dead throughout the cochlea turn, while supporting cells were preserved. ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM; N=5 in each group. Source data provided as source data file. Scale bar: 10 μm. [Figure 9-1]Figure 9 shows data demonstrating robust regeneration of new HCs in a severe HC loss model in vivo. Figure 9A: Schematic illustrating the experimental procedure of C57BL / 6j mice treated with kanamycin / furosemide, small molecule combination (or vehicle) and HC induction with ad-Atoh1-mCherry in vivo. Figure 9B: Kana / Furo / VLFsiFsiM (or vehicle) / ad-Atoh1-mCherry treated adult wild-type mouse cochlea samples labeled with ESPN / mCherry. In the control group without cocktail (VLFsiFsiM) treatment, few remaining outer hair cells were observed. In contrast, robust hair cell regeneration was observed throughout the cochlear turn after cocktail treatment. D: Dox; L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. **p<0.01, ***p<0.001, ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM; N=5–6 in each group. Source data are provided as source data file. Scale bar: 10 μm. [Figure 9-2] Data showing robust regeneration of new HCs in a severe HC reduction model in vivo. Figure 9C: Quantification and comparison showed a significant increase in the number of hair cells in cocktail-treated inner ears compared to vehicle-treated ears in the outer hair cell (OHC) region, across the cochlear turn. In the inner hair cell (IHC) region, IHC counts are unchanged due to lack of ad-Atoh1-mCherry infection and survival of IHCs. D: Dox; L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. **p<0.01, ***p<0.001, ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM; N=5-6 in each group. Source data provided as source data file. Scale bar: 10 µm. [Figure 10]Data showing new HCs in vivo at low magnification. Fig. 10A: Schematic illustrating the experimental procedure for C57BL / 6j mice treated with kanamycin / furosemide, small molecule combination (or vehicle), and HC induction with ad-Atoh1-mCherry in vivo. Fig. 10B: Apex to apex-mid samples of Kana / Furo / VLFsiFsiM (or vehicle) / ad-Atoh1-mCherry treated adult wild-type mouse cochlea labeled with ESPN / mCherry. D: Dox; L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. Scale bar: 20 μm. [Figure 11-1] Figure 11 shows data demonstrating robust regeneration of new HCs by supporting cell transdifferentiation in a severe HC loss model in vivo. Figure 11A: Schematic illustrating the experimental procedure of C57BL / 6j mice treated with kanamycin / furosemide, small molecule combinations (or vehicle) and HC induction by ad-Atoh1-mCherry in vivo. Figure 11B: Kana / Furo / VLFsiFsiM / ad-Atoh1-mCherry treated adult wild type mouse cochlear samples (arrowheads) stained with ESPN / SOX2 / mCherry. Double labeling of ESP-SOX2 is indicative that hair cells (MYO7A) are derived from supporting cells (SOX2) transdifferentiation. Figure 11C: Quantification and comparison showed a reduction in non-transdifferentiated SCs (ESPN- / SOX2+) as a result of supporting cell transdifferentiation into hair cells after cocktail treatment compared to freshly amputated ears. ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM; N=5 in each group. Scale bar: 20 μm. [Figure 11-2]Data showing robust regeneration of new HCs by supporting cell transdifferentiation in an in vivo severe HC loss model. (C) Quantification and comparison showed a reduction in non-transdifferentiated SCs (ESPN- / SOX2+) as a result of supporting cell transdifferentiation into hair cells after cocktail treatment compared to freshly amputated ears. ****p<0.0001, two-tailed unpaired Student's t-test. Error bars, mean ± SEM; N=5 in each group. Scale bar: 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0024] Detailed Description Hair cell loss is the main cause of permanent hearing loss in humans. Strategies to overcome irreversible cochlear hair cell damage and loss in mammals are crucial for hearing restoration. In the mature mammalian cochlea, simultaneous activation of c-Myc and Notch1 reprograms supporting cells and promotes hair cell regeneration. Given the important role of Myc in cell cycle, cell growth and stem cells, modulation of Myc with clinically relevant approaches is a priority in hair cell regeneration in the inner ear. We have shown that Myc is crucial in hair cell regeneration. However, it is difficult to activate Myc using small molecules, and MYC protein is unstable and difficult to produce. Therefore, we focused on identifying methods to activate Myc to sufficient levels for hair cell regeneration. We additionally screened for multiple targets, including HDAC inhibitors and inner ear developmental pathways, to identify those that act with Myc to efficiently regenerate hair cells in vitro and in vivo.

[0025] As described herein, a combination of small molecules and inhibitory nucleic acids was used to regenerate hair cells in adult wild-type mice. The original supporting cells of the regenerated hair cells were confirmed by lineage tracing. The regenerated hair cells were labeled with multiple mature hair cell markers, including SLC26A5 (prestin) for outer hair cells and SLC17A8 (VGLUT3) for inner hair cells. The new hair cells form connections with adult spiral ganglion neurons and take up FM1-43 dye, an indicator of the presence of the transduction complex. In addition, efficient hair cell regeneration in a mouse model of severe hair cell loss in vivo was demonstrated. In summary, the experimental results described herein identify a combinatorial approach using small molecules and inhibitory nucleic acids to regenerate hair cells in the wild-type adult mammalian cochlea, laying the foundation for hearing restoration using a clinically relevant approach.

[0026] Strategies for HC regeneration to restore hearing function are being actively explored. Multiple lines of evidence suggest that HC regeneration is possible in mammals. Spontaneous HC regeneration occurs in lower vertebrates such as birds and fish. Embryonic and neonatal mouse cochleae also retain the ability to regenerate HCs by enhancing the expression of certain genes essential for HC development. Hair cell regeneration in neonatal rodents was achieved by altering various signaling pathways. Activating sonic hedgehog signaling led to both cell cycle reentry and HC regeneration in the cochlear sensory epithelium. The ERBB2 pathway was also involved in promoting supporting cell proliferation and increasing MYO7A+ cell generation in neonatal mice. 12 However, this ability to generate new HCs declines rapidly after 2 weeks of age, even after altering the expression of HC regeneration signaling pathways. 9 However, none of the above treatments was sufficient to regenerate cochlear HCs in adult mice.

[0027] We have previously shown that transient coactivation of Myc and NICD (Notch1 intracellular domain) can successfully reprogram the adult mouse cochlea to a relatively young stage, with the generation of large numbers of new HCs after Atoh1 overexpression both in vitro and in vivo, restoring progenitor cell potential. 16 However, this "achievement" occurs only in transgenic animals that are not amenable to clinical trials in human patients. Thus, HC regeneration in the human cochlea remains particularly challenging.

[0028] By screening various combinations of small molecules and inhibitory nucleic acids as described herein, unique combinations of agents were identified that can regulate Myc and reprogram well-differentiated cochlear epithelial cells, including Sox2+ supporting cells (SCs), to regain progenitor capacity and robustly regenerate new HCs in adults. Furthermore, by comparing newly regenerated HCs from different HC-reduced mouse models, it was revealed that different reprogramming approaches in the adult cochlea lead to different maturation levels of new HCs. Thus, the experimental data provided herein demonstrate that robust regeneration of cochlear HCs can be achieved in adult mice using clinically relevant approaches.

[0029] Thus, the present disclosure provides, in some aspects, methods for regenerating hair cells in the adult wild-type cochlea and utricle using unique combinations of agents.

[0030] I. Agents for regenerating hair cells (HCs) in the adult mammalian cochlea Provided herein is a method for regenerating hair cells in the cochlea of ​​an adult mammal, comprising: (1) a reprogramming step, in which the adult mammalian cochlea is successfully reprogrammed to a relatively young stage, thereby allowing the cochlea to regain progenitor cell potential; and (2) a transdifferentiation step, in which activation of an HC fate determining factor (Atoh1) in the reprogrammed adult progenitor cells results in HC regeneration.

[0031] In the reprogramming step described herein, the mammalian cochlea is reprogrammed to a less differentiated state using a unique combination of agents including an HDAC inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or a combination thereof. In some embodiments, the unique combination of agents for use in the methods described herein also includes a Wnt agonist and / or a cAMP agonist.

[0032] As used herein, "drug" refers to any molecule (e.g., small molecule, nucleic acid) that can reprogram mammalian cochlea for hair cell regeneration and / or regenerate hair cells in mammalian cochlea.Drugs for use in the described methods include HDAC inhibitors, inhibitory nucleic acids that target Fir, Mxi1, Fbxw7 or combinations thereof, Wnt agonists, cAMP agonists and Atoh1 activators.

[0033] (a) Histone deacetylase (HDAC) inhibitors The method described herein comprises the use of HDAC inhibitor to reprogram adult mammalian cochlea to less differentiated progenitor cell state.In some embodiments, HDAC inhibitor is HDAC class I inhibitor, HDAC class II inhibitor, HDAC class III inhibitor, and / or pan-HDAC inhibitor.In some embodiments, HDAC class III inhibitor is SIRT1 inhibitor and / or SIRT2 inhibitor.

[0034] As used herein, "HDAC inhibitor" refers to a compound that binds to and inhibits one or more HDACs, thereby affecting the enzymatic activity of HDACs. HDAC refers to any one of a family of enzymes that catalyze the removal of acetyl groups from the ε-amino groups of lysine residues at the N-terminus of histones. Unless otherwise indicated by context, the term "histone" refers to any histone protein, including HI, H2A, H2B, H3, H4 and H5, from any species. Human HDAC proteins are classified into four classes: class I includes HDAC1, HDAC2, HDAC3 and HDAC8; class II includes HDAC4, HDAC5, HDAC7 and HDAC9; class III includes SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7; and class IV includes HDAC11. HDAC inhibitors may be pan-HDAC inhibitors or may show selectivity for one or more HDACs.

[0035] In some embodiments, the HDAC inhibitor is sodium butyrate, trichostatin A, hydroxamic acid, cyclic tetrapeptide, trapoxin B, depsipeptide, benzamide, electrophilic ketone, fatty acid compound, pyroxamide, phenylbutyrate, valproic acid, hydroxamic acid, romidepsin, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (L BH589), entinostat (MS275), CI-994 (N-acetylglutaminidine, also tacedinaline), entinostat (SNDX-275; formerly MS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, gibinostat (ITF2357), mocetinostat (MGCD0103), or combinations thereof. Examples of HDAC inhibitors include, but are not limited to, the compounds listed in Table 1.

[0036] [Table 1-1]

[0037] [Table 1-2]

[0038] [Table 1-3]

[0039] (b) Inhibitory nucleic acids targeting Fir, Mxi1 and Fbxw7 Embodiments of the present disclosure provide methods for reprogramming the adult mammalian cochlea to a less differentiated progenitor cell state using one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or combinations thereof.

[0040] The sequences of human Fir, human Mxil and human Fbxw7 are known in the art. For example, the sequence of human Fir is available in the NCBI database as NM_078480.3 (SEQ ID NO: 1). For example, the sequence of human Mxil is available in the NCBI database as NM_005962.5 (SEQ ID NO: 2). For example, the sequence of human Fbxw7 is available in the NCBI database as NM_033632.3 (SEQ ID NO: 3). See Table 2 below.

[0041] [Table 2]

[0042] As used herein, "inhibitory nucleic acid" refers to a nucleic acid or a mimic thereof that, when administered to a mammalian cell, results in a reduction in the expression of a target gene. Typically, an inhibitory nucleic acid comprises at least a portion of a target nucleic acid molecule that hybridizes to at least a portion of the target nucleic acid and regulates its function. In some embodiments, the expression of the target gene is reduced by at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% or more.

[0043] Examples of inhibitory nucleic acid include, but are not limited to, short interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotide.Inhibitory nucleic acid can include DNA, RNA, modified nucleic acid and combinations thereof (e.g., DNA / RNA hybrid).Methods for making and delivering inhibitory nucleic acid that targets specific sequences are known in the art. See, e.g., Ramachandran and Ignacimuthu, Appl Biochem Biotechnol. 2013; 169(6):1774-89; Li et al., J Control Release 2013; 172(2):589-600; Lochmatter and Mullis, Horm Res Paediatr. 2011; 75(1):63-9; Higuchi et al., BioDrugs. 2010; 24(3):195-205; and Ming et al., Expert Opin Drug Deliv. 2011; 8(4):435-49.

[0044] The methods described herein encompass the use of a single inhibitory nucleic acid or multiple inhibitory nucleic acids.For example, when targeting Fir and Mxi1, the methods and compositions described herein can include the use of a single inhibitory nucleic acid that targets Fir and Mxi1, or the use of an inhibitory nucleic acid that targets Fir and an inhibitory nucleic acid that targets Mxi1.

[0045] In some embodiments, the inhibitory nucleic acid targets one of Fir, Mxil or Fbxw7 (e.g., the inhibitory nucleic acid targets Fir, the inhibitory nucleic acid targets Mxil, or the inhibitory nucleic acid targets Fbxw7). In some embodiments, the inhibitory nucleic acid targets two of Fir, Mxil and Fbxw7 (e.g., the inhibitory nucleic acid targets Fir and Mxil, the inhibitory nucleic acid targets Fir and Fbxw7, or the inhibitory nucleic acid targets Mxil and Fbxw7). In some embodiments, the inhibitory nucleic acid targets each of Fir, Mxil and Fbxw7.

[0046] (c) Wnt agonists and cAMP agonists The methods described herein, in some embodiments, comprise the use of Wnt agonists and / or cAMP agonists to reprogram the adult mammalian cochlea to a less differentiated progenitor cell state.

[0047] As used herein, the term "Wnt agonist" refers to any agent that activates the Wnt / β-catenin pathway or inhibits the activity and / or expression of an inhibitor of Wnt / β-catenin signaling, e.g., an antagonist or inhibitor of GSK-3β activity.

[0048] Wnt agonists include Wnt proteins or other compounds that directly bind to Frizzled and lipoprotein receptor-related protein 5 / 6 (LRP5 / 6) co-receptor proteins (e.g., Frizzled receptor activators, LRP5 / 6 activators) in a manner that promotes increased concentration of β-catenin in the nucleus of mammalian cells. Alternatively, Wnt agonists can function by inhibiting one or more secreted Frizzled-related proteins (sFRPs) (e.g., sFRP inhibitors) or Wnt inhibitory proteins (WIFs) (e.g., WIF inhibitors), which bind to Wnt proteins and sequester them from interacting with endogenous Wnt co-receptors. Examples of Wnt agonists also include, but are not limited to, glycogen synthase kinase-3β (GSK-3β) inhibitors, Wnt activators, Dishevelled (Dvl) activators, Axin inhibitors, Dickkopf (Dkk) inhibitors and Groucho inhibitors. GSK-3β is a kinase that complexes with Axin, APC (Adenomatous polyposis coli) and β-catenin to prepare β-catenin for downstream degradation by the proteasome. Dishevelled (Dvl) is an intracellular protein that relays signals from activated Notch receptors to downstream effectors. Dvl is recruited by the receptor Frizzled and prevents constitutive descruction of β-catenin. Dkk is a secreted protein that acts to isolate LRP5 / 6 co-receptor proteins, thereby inhibiting Wnt signaling. Groucho is a protein that complexes with TLE in the nucleus to repress gene expression. When β-catenin enters the nucleus, it disrupts the Groucho / TLE complex and activates gene expression. Wnt agonists can be small molecule compounds that activate Wnt signaling. See, for example, International Publication No. WO2018172997, the relevant disclosures of which are incorporated by reference for purposes and subject matter referenced herein. Examples of Wnt agonists include, but are not limited to, the compounds listed in Table 3.

[0049] [Table 3-1]

[0050] [Table 3-2]

[0051] As used herein, "cAMP agonist" refers to an agent that increases the intracellular level of cAMP compared to the background physiological intracellular level in the absence of the agent. Examples of cAMP agonists include, but are not limited to, forskolin, rolipram, NKH477, PACAP1-27, PACAP1-38 and isoproterenol.

[0052] (d) Atoh1 activator After reprogramming the adult wild-type cochlea to a less differentiated state, Atoh1 activity can be increased to regenerate HCs in the adult mammalian cochlea. Thus, the methods described herein, in some embodiments, include the use of an agent that increases Atoh1 activity to regenerate cochlear hair cells in adult wild-type cochleae that have been reprogrammed to a less differentiated state (e.g., reprogrammed to re-express inner ear progenitor cell genes).

[0053] As used herein, the term "Atoh1" refers to a protein that belongs to the basic helix-loop-helix (BHLH) family of transcription factors involved in the formation of hair cells in the mammalian inner ear.

[0054] Any method for increasing Atoh1 activity can be used in the methods described herein. Methods for increasing Atoh1 activity (including the use of Atoh1 agonist) are known in the art (see, for example, U.S. Patent No. 8,188,131; U.S. Patent Application Publication No. 20110305674; U.S. Patent Application Publication No. 20090232780; Kwan et al. (2009) INT'L SYMPOSIUM ON OLFACTION AND TASTE: ANN. NY ACAD. SCI. 1170:28-33; Daudet et al. (2009) Dev. Bio. 326:86-100; Takebayashi et al. (2007) Dev. Bio. 307:165-178; and Ahmed et al. (2012) Dev. Cell 22(2):377-390).

[0055] Non-limiting examples of methods for increasing Atoh1 activity that can be used in the methods described herein are provided in U.S. Patent Application Publication No. 2020 / 0338160, which is incorporated by reference in its entirety herein.

[0056] The agent for increasing Atoh1 activity can be the nucleic acid encoding Atoh1, Atoh1 protein or Atoh1 agonist.Exemplary Atoh1 polypeptide includes, for example, NP_005163.1, which is referred to in NCBI protein database.Exemplary Atoh1 nucleic acid sequence that can be expressed in target cell includes, for example, NM_005172.1, which is referred to in NCBI gene database.

[0057] Other exemplary Atoh1 activators described in U.S. Pat. No. 8,188,131 include 4-(4-chlorophenyl)-1-(5H-pyrimido[5,4-b]indol-4-yl)-1H-pyrazol-3-amine; 6-chloro-1-(2-chlorobenzyloxy)-2-phenyl-1H-benzo[d]imidazole; 6-chloro-1-(2-chlorobenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole; 6-chloro-2-(4-methoxyphenyl)-1-(4-methylbenzyloxy)-1H-benzo [d]imidazole;6-chloro-1-(3,5-dimethylbenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole;6-chloro-1-(4-methoxybenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole;1-(4-methylbenzyloxy)-6-nitro-2-phenyl-1H-benzo[d]imidazole;4-(1H-benzo[d]imidazol-2-yl)phenol;2,5-dichloro-N-((1-methyl-H-benzo[d]imidazol-2-yl)methyl)aniline;4 -(2-(1-Methyl-1H-benzo[d]imidazol-2-yl)ethyl)aniline;2-((2-methoxyphenoxy)methyl)-1H-benzo[d]imidazole;2-((4-fluorophenoxy)methyl)-1-methyl-1H-benzo[d]imidazole;2-(phenylthiomethyl)-1H-benzo[d]imidazole;3-(6-Methyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-imine;N-(2-(1H-benzo[d]imidazol-2-yl)phenyl)isobutyramide;2-(o-Tolyloxy) 2-(4-Methoxyphenyl)-1-phenethyl-1H-benzo[d]imidazole;N-(6-Bromobenzo[d]thiazol-2-yl)thiophene-2-carboxamide;N-(benzo[d]thiazol-2-yl)-1-methyl-1H-pyrazole-5-carboxamide;2-(4-Fluorobenzylthio)benzo[d]thiazole;5-Chloro-N-methylbenzo[d]thiazol-2-amine;N-(6-acetamidobenzo[d]thiazol-2-yl)furan-2-carboxamide;N-(6-fluorobenzo[d]thiazol-2-yl)-3-methoxybenzamide;2-(benzo[d]oxazol-2-ylthio)-N-(2-chlorophenyl)acetamide;5-chloro-2-phenylbenzo[d]oxazole;5-methyl-2-m-tolylbenzo[d]oxazole;2-(4-isobutoxyphenyl)-3-(naphthalen-2-yl)-2,3-dihydroquinazolin-4(1H)-one;N-(2-(2-(4-fluorophenyl)-2-oxoethylthio)-4-oxoquinazolin-3(4H)-yl)benzamide; 2-(4-Chlorophenyl)-4-(4-methoxyphenyl)-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine;2-(3-pyridyl)-4-(4-bromophenyl)-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine;N-sec-butyl-1,7,7-trimethyl-9-oxo-8,9-dihydro-7H-furo[3,2-f]chromene-2-carboxamide;N-(3-carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzofuran-2-carboxamide;3-Chloro -N-(5-chloropyridin-2-yl)benzo[b]thiophene-2-carboxamide;3-chloro-N-((tetrahydrofuran-2-yl)methyl)benzo[b]thiophene-2-carboxamide;N-(3-(5-chloro-3-methylbenzo[b]thiophene-2-yl)-1H-pyrazol-5-yl)acetamide;2-(naphthalen-2-yl)-1H-indole;2-(pyridin-2-yl)-1H-indole;N-(2-chlorophenyl)-2-(1H-indol-3-yl)-2-oxoacetamide;2-m-tolylquinoline ;2-(4-(2-methoxyphenyl)piperazin-1-yl)quinolone;2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(2,3-dihydro-1H-inden-2-yl)acetamide;1-phenethyl-1H-benzo[d][1,2,3]triazole;7-(4-fluorobenzyloxy)-2H-chromen-2-one;N-(2,4-dichlorophenyl)-8-methoxy-2H-chromene-3-carboxamide;N-(3-chlorophenyl)-8-methyl-3,4-dihydroquinoline-1(2H)-carbothioamide;7-Methoxy-5-methyl-2-phenyl-4H-chromen-4-one;2-(3,4-dimethylphenyl)quinoxaline;4-Bromo-N-(5-chloropyridin-2-yl)benzamide;3-Amino-6,7,8,9-tetrahydro-5H-cyclohepta[e]thieno[2,3-b]pyridine-2-carboxamide;(Z)-3-Methyl-N'-(nicotinoyloxy)benzimidamide;N,N-Diethyl-6-methoxythieno[2,3-b]quinoline-2-carboxamide;6-(4-Methoxyphenyl)-1,2,3,4-tetrahydro -1,5-Naphthyridine;5-Bromo-N-(2-(phenylthio)ethyl)nicotinamide;N-(6-Methylpyridin-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide;2-(4-Methylbenzylthio)oxazolo[4,5-b]pyridine;N-(2-Methoxyethyl)-5-p-tolylpyrimidin-2-amine;4-(5-(Benzo[b]thiophen-2-yl)pyrimidin-2-yl)morpholino;4-(5-(4-Fluorophenyl)pyrimidin-2-yl)morpholino;N-(4-Bromo-3-methyl N-(4-Methoxyphenyl)quinazolin-4-amine;N-(3-Methoxyphenyl)-9H-purin-6-amine;N,N-Diethyl-1-m-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;(5-(4-Bromophenyl)furan-2-yl)(morpholino)methanone;(Z)-4-Bromo-N'-(furan-2-carbonyloxy)benzimidamide;N-(4-Iodophenyl)furan-2-carboxamide;5-(5-(2,4-difluorophenyl)furan-2-yl)-1- (Methylsulfonyl)-1H-pyrazole;1-(3-amino-5-(4-tert-butylphenyl)thiophen-2-yl)ethanone;N-(3-cyano(cayano)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-fluorobenzamide;N-(5-chloropyridin-2-yl)thiophene-2-carboxamide;N-(2-(4-fluorophenoxy)ethyl)thiophene-2-carboxamide;2,5-dimethyl-N-phenyl-1-(thiophen-2-ylmethyl)-1H-pyrrole-3-carboxamide;N-(3-cyanothiophen-2-yl)-4-isopropoxybenzamide;2-(4-methoxyphenoxy)-N-(thiazol-2-yl)acetamide;4-(4-methoxyphenyl)-N-(3-methylpyridin-2-yl)thiazol-2-amine;4-(biphenyl-4-yl)thiazol-2-amine;4-(4-(4-methoxyphenyl)thiazol-2-yl)-3-methylisoxazol-5-amine;N-(2-methoxyphenyl)-4-phenylthiazol-2-amine;1-(4-amino-2-(m-tolylamino)thiazo 4-(4-Chlorophenyl)-1-(5H-pyrimido[5,4-b]indol-4-yl)-1H-pyrazol-3-amine;2-(4-Chlorophenyl)-6-ethyl-5-methylpyrazolo[1,5-a]pyrimidin-7(4H)-one;5-Methoxy-2-(5-phenyl-1H-pyrazol-3-yl)phenol;(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methanol;N-(2,5-dichlorophenyl)-1-ethyl-1H-pyrazol-3-carbo 4-Chloro-1-methyl-N-(2-oxo-2-phenylethyl)-1H-pyrazole-3-carboxamide;N-(3-(5-tert-butyl-2-methylfuran-3-yl)-1H-pyrazol-5-yl)benzamide;N-(5-methylisoxazol-3-yl)benzo[d][1,3]dioxole-5-carboxamide;(5-(4-bromophenyl)isoxazol-3-yl)(morpholino)methanone;N-(4-bromophenyl)-5-isopropylisoxazole-3-carboxamide;5-((4-chloro-2- 5-(2-Methoxyphenyl)-3-p-tolyl-1,2,4-oxadiazole;5-(phenoxymethyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;5-(2-Chloro-4-methylphenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;3-(2-Chlorophenyl)-5-p-tolyl-1,2,4-oxadiazole;5-(piperidin-1-ylmethyl)-3-p-tolyl-1,2,4-oxadiazole;5-(4-bromophenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;5-(2-bromophenyl)-3-(4-bromophenyl)-1,2,4-oxadiazole;5-(2-bromo-5-methoxyphenyl)-3-(thiophenyl-2-yl)-1,2,4-oxadiazole;3-(2-fluorophenyl)-N-(3-(piperidin-1-yl)propyl)-1,2,4-oxadiazole-5-amine;2-(2-chlorobenzoyl)-N-(4-fluorophenyl)hydrazinecarbothioamide;2-(methylamino)-N-phenethylbenzamide;4-tert-butyl-N-((tetrahydrofuran-2-yl)methyl)benzamide;2-phenyl-5-o-tolyl-1,3,4-oxadiazole;4-(3-(4-chlorophenyl)-4,5-dihydro-1 H-1,2,4-triazol-5-yl)-N,N-dimethylaniline;7-Methoxy-2-(4-methoxyphenyl)-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazine-5,1'-cyclohexane];6-Oxo-2-(4-(3-(trifluoromethyl)phenoxy)phenyl)-1,4,5,6-tetrahydropyridine-3-carbonitrile;6-(4 2-(2-bromophenoxy)-N-(4H-1,2,4-triazol-3-yl)acetamide; 1-(indolin-1-yl)-2-phenoxyethanone; 2-(4-chlorophenyl)-6,7,8,9-tetrahydrobenzo[e]imidazo[1,2-b][1,2,4]triazine; and pharma- ceutically acceptable salts thereof.

[0058] (e) Pharmaceutical Composition Any of the compounds for regenerating cochlear hair cells described herein can be mixed with a pharma- ceutically acceptable carrier or excipient to form a pharmaceutical composition for use in the methods of producing a population of cochlear progenitor cells and in the methods of treating hearing loss.

[0059] The pharmaceutical composition comprising one or more compounds described herein can be formulated according to the intended method of administration.For example, the pharmaceutical composition can be formulated for local or systemic administration, for example, ear drops (e.g., ear drops) or injection, insufflation (e.g., into the ear), intravenous, topical or oral administration.One or more compounds described herein can be formulated as pharmaceutical compositions for direct administration to subjects.

[0060] The nature of the pharmaceutical composition for administration depends on the mode of administration and can be easily determined by those skilled in the art. In some embodiments, the pharmaceutical composition is sterile or sterilizable. The therapeutic composition of interest in the present invention may contain carriers or excipients, many of which are known to those skilled in the art. Excipients that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water and glycerol. Nucleic acids, polypeptides, small molecules and other regulatory compounds of interest in the present invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous or oral.

[0061] Pharmaceutical compositions can be formulated in various ways depending on the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for oral ingestion; gels or powders can be made for oral ingestion or topical application. Methods for making such formulations are well known and are described, for example, in Remington: The Science and Practice of Pharmacy, 22 nd Ed., Allen, ed., Mack Publishing Co., Easton, Pa., 2012.

[0062] One or more compounds may be administered, for example, as a pharmaceutical composition, directly and / or locally by injection or through surgical placement, for example, in the inner ear. The amount of the pharmaceutical composition may be described as an effective amount or as a therapeutically effective amount. If application over a period of time is appropriate or desired, the composition of the present invention may be placed in a sustained release formulation or in an implantable device (e.g., a pump).

[0063] Alternatively or additionally, the pharmaceutical composition can be formulated for systemic parenteral administration by injection, for example, by bolus injection or continuous infusion. Such formulations can be in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulatory agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0064] In addition to the formulations already described, the composition may be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously). Thus, for example, the composition may be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or a sparingly soluble derivative, such as, for example, a sparingly soluble salt.

[0065] Pharmaceutical compositions formulated for systemic oral administration may take the form of tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

[0066] In some embodiments, the pharmaceutical compositions described herein may include one or more compounds formulated by any of the methods described herein.

[0067] II. Methods for reprogramming the mammalian cochlea for hair cell regeneration Aspects of the present disclosure provide a method for reprogramming an adult mammalian cochlea, comprising contacting the mammalian cochlea with an HDAC inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or a combination thereof, under conditions and for a time sufficient to produce a population of cochlear progenitor cells. In some embodiments, the method described herein further comprises contacting the mammalian cochlea with a Wnt agonist (e.g., LiCl) and / or a cAMP agonist (e.g., forskolin).

[0068] As used herein, the term "reprogramming" refers to the process of changing or returning the differentiation state of cochlear cells. For example, an adult mammalian cochlea containing differentiated cochlear cells can be reprogrammed so that the differentiated cochlear cells are transformed into cochlear progenitor cells. Reprogramming encompasses the complete or partial return of the differentiation state of cochlear cells. In some embodiments, reprogramming renders the mammalian cochlea or mammalian cochlear cells more susceptible to hair cell regeneration when contacted with an Atoh1 activator.

[0069] As used herein, the term "progenitor cell" refers to an immature or undifferentiated cell that has the potential to mature (differentiate) into a specific cell type, for example, a hair cell. As used herein, "cochlear progenitor cell" refers to a progenitor cell that has the differentiation potential to form cochlear hair cells. Progenitor cells have a cellular phenotype that is more primitive than the cells that can be generated by differentiation. For example, cochlear progenitor cells can express progenitor cell markers such as Six1, Eya1, Gata3, Sox2, Notch1, Hes5, or a combination.

[0070] In order to carry out the reprogramming method described herein, the cochlea of ​​an adult mammal is contacted with a combination of drugs, such as HDAC inhibitor and one or more inhibitory nucleic acids.As used herein, the term "contacting" refers to the exposure of the cochlea of ​​a mammal to a combination of drugs for hair cell regeneration (e.g., HDAC inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7 or a combination thereof, and optionally a Wnt agonist and / or a cAMP agonist) under conditions for producing a population of cochlear progenitor cells in the cochlea of ​​a mammal, and for a time sufficient for the production thereof.The cochlea of ​​a mammal can be contacted with a combination of drugs for hair cell regeneration in vitro (e.g., in culture) or in vivo (e.g., in a subject).

[0071] The methods described herein include contacting a mammalian cochlea with a combination of agents described herein for a period of time suitable to reprogram cochlear cells to a less differentiated progenitor cell state. In some embodiments, the methods described herein include contacting a mammalian cochlea with a combination of agents described herein for 1-15 days, e.g., 2-15 days, 3-15 days, 4-15 days, 5-15 days, 6-15 days, 7-15 days, 8-15 days, 9-15 days, 10-15 days, 11-15 days, 12-15 days, 13-15 days, 14-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, or 1-2 days.

[0072] The presence of cochlear progenitor cells can be determined using any method known in the art, for example, by detecting the expression of one or more progenitor cell genes, such as Six1, Eya1, Gata3, Sox2, Notch1, Hes5, or combinations thereof, in the mammalian cochlea.

[0073] III. Treatment Methods Provided herein is a method for treating hearing loss in mammalian subjects (e.g., human or non-human veterinary subjects) using HDAC inhibitors and one or more inhibitory nucleic acids that target Fir, Mxi1, Fbxw7 or combinations thereof, and optionally Wnt agonist and / or cAMP agonist.In some embodiments, the subject is a subject after neonatal age (e.g., pediatric, adolescent or adult, e.g., over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 years old).The subject can be treated with the combination of drugs described herein.

[0074] The methods described herein encompass treating hearing loss and any disorder that occurs as a result of cochlear hair cell loss, such as noise-induced permanent hearing loss, drug-induced hearing loss, age-related hearing loss, vestibular dysfunction, and the like.

[0075] In some embodiments, the hearing loss is sensorineural hearing loss, which may result from damage or dysfunction of the cochlea, for example, loss or damage to the sensory epithelium resulting in a loss of hair cells.

[0076] In some embodiments, hearing loss can be due to any reason or can be the result of any type of event.For example, due to genetic or congenital defects; for example, a human subject may be deaf from birth, and may be deaf or have reduced hearing as a result of gradual loss of hearing due to genetic or congenital defects.In another example, hearing loss can be the result of traumatic events, such as physical trauma to the ear structure, or sudden loud noise, or long-term exposure to loud noise.For example, long-term exposure to concert venues, airport runways, and construction areas can cause inner ear damage and subsequent hearing loss.

[0077] In some embodiments, the hearing loss may be due to chemically induced toxic hearing loss, where ototoxins include anti-cancer drugs, salicylates, therapeutic drugs including quinine and aminoglycoside antibiotics, contaminants in food or medicine, and environmental or industrial pollutants. In some embodiments, the hearing loss may result from aging.

[0078] In some embodiments, the method includes selecting a subject. Suitable subjects for treatment include those who have or are at risk of having a decrease in inner ear hair cells, or those who have or are at risk of having sensorineural hearing loss. Any subject who has experienced or is at risk of developing hearing loss is a candidate for the treatment method described herein. In some examples, the subject has noise-induced permanent hearing loss, drug-induced hearing loss, age-related hearing loss, sudden sensorineural hearing loss, hearing loss due to viral infection, tinnitus, vestibular dysfunction, or a combination thereof. Human subjects who have or are at risk of developing hearing loss may hear less well than the average human, or may hear less well than a human before experiencing hearing loss. For example, hearing may be impaired by at least 5, 10, 30, 50% or more.

[0079] In some embodiments, the methods include administering to a subject a combination of agents described herein within 1, 2, 3, 4, 5, 6, or 7 days or 1, 2, 3, 4, 5, or 6 weeks of exposure to an ototoxic insult, e.g., a physical (noise, trauma) or chemical (ototoxin) insult that results or may result in hair cell loss.

[0080] In some embodiments, subjects suitable for treatment using the agents and methods featured in the present invention may include subjects with vestibular dysfunction, including bilateral and unilateral vestibular dysfunction; the method includes administering a therapeutically effective amount of an agent described herein, for example, by systemic administration or administration via the endolymphatic sac (ES). Vestibular dysfunction is an inner ear dysfunction characterized by symptoms including dizziness, imbalance, vertigo, nausea and fuzzy vision, and may be accompanied by hearing problems, fatigue and changes in cognitive function. Vestibular dysfunction that can be treated by the methods described herein may be the result of genetic or congenital defects that result in a reduction in vestibular hair cells; infection, viral or bacterial infection, etc.; or injury, traumatic or non-traumatic injury, etc. In some embodiments, balance disorders or Meniere's disease (idiopathic endolymphatic hydrops) can be treated by the methods described herein. Vestibular dysfunction is most commonly examined by measuring individual symptoms of the disorder (e.g., dizziness, nausea and fuzzy vision).

[0081] Alternatively or additionally, the agents and methods featured in the present invention may be used prophylactically, such as to prevent, reduce, or slow the progression of hearing loss, hearing loss, or other hearing impairments associated with hair cell loss. For example, a composition containing one or more agents may be administered in conjunction with (e.g., before, after, or simultaneously with) an ototoxic treatment, i.e., a treatment that carries a risk of hair cell toxicity and thereby causing hearing loss. Ototoxic drugs include antibiotics, neomycin, kanamycin, amikacin, viomycin, gentamicin, tobramycin, erythromycin, vancomycin and streptomycin; chemotherapy drugs, such as cisplatin; nonsteroidal anti-inflammatory drugs (NSAIDs), such as choline magnesium trisalicylate, diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate, sulindac and tolmetin; diuretics; salicylates, such as aspirin; and certain malaria treatments, such as quinine and chloroquine. For example, subjects undergoing chemotherapy can be treated using the agents and methods described herein. For example, the chemotherapy drug cisplatin is known to cause hearing loss. Thus, compositions containing one or more agents can be administered together with (e.g., before, after, or simultaneously with) cisplatin treatment to prevent or reduce the severity of cisplatin side effects. Such compositions can be administered before, after, and / or simultaneously with a second therapeutic agent. The two agents can be administered by the same route or by different routes.

[0082] In general, the agents and methods described herein can be used to cause hair cell growth and / or increase the number of hair cells in the inner ear (e.g., the cochlea and / or the utricle).For example, the number of hair cells in the inner ear (e.g., the cochlea and / or the utricle) can be increased by about 2, 3, 4, 6, 8 or 10 times or more compared to the number of hair cells before treatment.This new hair cell growth can effectively restore the subject's ability to hear, or establish at least partial improvement.For example, administration of the agent can improve hearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

[0083] In some cases, the composition can be administered to a subject, for example, a subject that is identified as needing treatment, by using a systemic administration route.Systemic administration routes include, but are not limited to, parenteral administration routes, for example, intravenous injection, intramuscular injection and intraperitoneal injection; enteral administration routes, for example, oral route, administration by lozenges, compressed tablets, pills, tablets, capsules, drops (for example, ear drops), syrups, suspensions and emulsions; transdermal administration routes; and inhalation (for example, nasal drops).

[0084] In some cases, the compositions can be administered to a subject, e.g., a subject identified as in need of treatment, using systemic or local routes of administration. Such routes of local administration include administering one or more agents to the subject's ear and / or the subject's inner ear, e.g., by injection and / or using a pump.

[0085] In some cases, the composition can be injected into the ear (e.g., auricular administration), into the lumen of the cochlea (e.g., scala media, Sc vestibulae and Sc tympani), etc. For example, the composition can be administered by intratympanic injection (e.g., into the middle ear), intracochlear delivery (e.g., stapes base), and / or injection into the outer, middle and / or inner ear. Such methods are routinely used in the art, for example, for administration of steroids and antibiotics to the human ear. The injection can be, for example, through the cochlea window of the ear or through the cochlea capsule. In another exemplary mode of administration, the composition can be administered in situ via a catheter or pump. The catheter or pump, for example, directs the pharmaceutical composition into the lumen of the cochlea or into the cochlea window of the ear. Exemplary drug delivery devices and methods suitable for administering one or more compounds to the ear, for example, to the human ear, are described by McKenna et al., (US Patent Publication No. 2006 / 0030837) and Jacobsen et al., (US Patent No. 7,206,639). In some embodiments, the catheter or pump may be placed in the ear (e.g., outer ear, middle ear and / or inner ear) of a subject, for example, during a surgical procedure. In some embodiments, the catheter or pump may be placed in the ear (e.g., outer ear, middle ear and / or inner ear) of a subject, for example, without the need for a surgical procedure.

[0086] In some cases, the disclosure includes treating a subject by administering to the subject cells produced using the agents and methods disclosed herein. Generally, such methods can be used to promote complete or partial differentiation of cells into or toward mature cell types of the inner ear (e.g., hair cells) in vitro. The cells resulting from such methods can then be transplanted or implanted into a subject in need of such treatment. The cell culture methods required to carry out these methods are described herein, including methods for identifying and selecting suitable cell types, methods for promoting complete or partial differentiation of selected cells, methods for identifying fully or partially differentiated cell types, and methods for implanting fully or partially differentiated cells. Suitable target cells for use in these methods are described above.

[0087] Administration of cells to a subject, whether alone or in combination with an agent disclosed herein, can include administration of undifferentiated, partially differentiated, and fully differentiated cells, including mixtures of undifferentiated, partially differentiated, and fully differentiated cells. As disclosed herein, cells that are less than fully differentiated may continue to differentiate toward fully differentiated cells after administration to a subject.

[0088] After treatment, if necessary, the subject may be tested for improvement in hearing or other symptoms related to inner ear disorders. Methods for measuring hearing are well known and include pure tone audiometry, air conduction and bone conduction tests. These tests measure the limits of sound pressure (intensity) and pitch (frequency) that the subject can hear. Human hearing tests include auditory behavioral response (for infants up to 7 months), visual reinforcement orientation audiometry (for children aged 7 months to 3 years); play audiometry for children over 3 years; and standard hearing tests for older children and adults, such as whispered speech, pure tone audiometry; tuning fork test; brainstem auditory evoked potential (BAER) test or auditory brainstem evoked potential (ABEP) test. Otoacoustic luminescence tests can be used to test the function of hair cells in the cochlea, and electrocochleography provides information about the function of the first part of the cochlea and the neural pathway to the brain. In some embodiments, treatment may be continued with or without the modification, or may be stopped. EXAMPLES

[0089] In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the methods and compositions provided herein and are not to be construed as limiting the scope thereof in any manner.

[0090] material and method The following materials and methods were used in the examples described herein.

[0091] Mouse model Rosa-rtTA (rtTA), Sox2Cre transgenic mice and Ai14 tdTomato reporter mice were purchased from Jackson Laboratory (Stock#006965;017593;007914); tet-on-Myc mice were from Dr. M. Bishop, University of California, San Francisco; tet-on-NICD mice were from Dr. D. Melton, Harvard University. For transgenic rtTA / tet-on-Myc / tet-on-NICD mice, the background was mixed C57 / 129SvJ / CD1 with approximately equal numbers of sexes; Atoh1-nGFP mice were from Dr. Jane Johnson (University of Texas Southwestern Medical Center, Dallas, TX); wild-type mice were C57BL / 6 from Jackson Laboratory. All experiments were performed in accordance with ethical regulations and approved by the Animal Care Committee of Massachusetts Eye and Ear and Harvard Medical School.

[0092] 3. In vitro Culture of Adult Cochleae and Viral Infection Unlike the neonatal cochlea culture method, where the cochlea was separated from the bone, whole cochleae of adult mice (4–6 weeks old) were cut with the bone attached. The bulla was first removed from the skull and immersed in 75% ethanol for 3 min before being placed in HBSS. The vestibular region was also removed. Under a dissecting microscope, the middle ear, blood vessels and debris were removed from the bulla. The bone covering the apical turn was removed, and the fenestra cochleae and oval window membranes were opened to allow medium exchange with cochlear fluid. Ligament parts and Reissner's membrane at both ends of the cochlea were also removed to facilitate access of the medium to the sensory epithelium region. The cochleae were maintained for 14–18 days in DMEM / F12 (Invitrogen) supplemented with N2 and B27 (both from Invitrogen). Ad-Atoh1 / ad-Atoh1-mCherry adenovirus was purchased from SignaGen Laboratories, Rockville, MD, and was administered at 6 × 10 10 The titer was pfu / ml.

[0093] In vitro reprogramming for HC regeneration In the rtTA / tet-Myc / tet-NICD mouse model, the cochleae were treated with Dox (Sigma, final concentration 2 μg / ml); in the Sox2 / tdT / rtTA / tet-Myc / tet-NICD mouse model, the cochleae were treated with Dox (Sigma, final concentration 2 μg / ml) and 4-hydroxytamoxifen (Tm, Sigma) (20 ng / ml); in the rtTA / tet-Myc and rtTA / tet-Myc / Atoh1-GFP mouse models, Cochleae were treated with Dox (Sigma, final concentration 2 μg / ml) and VPA (2 mM); in the Sox2 / tdT / rtTA / tet-Myc mouse model, cochleae were treated with 4-hydroxytamoxifen (20 ng / ml), Dox (Sigma, 2 μg / ml), and VPA (Sigma, 2 mM); in the rtTA / tet-NICD mouse model, cochleae were treated with Dox (Sigma, final concentration 2 μg / ml), LiCl (Sigma, 8 mM), FSK (Tocris Bioscience, 20 μM), siFIR (Santa Cruz Bio-technology, 0.02 μM), and siMxi1 (Santa Cruz Bio-technology, 0.02 μM). In the Sox2 / tdT / rtTA / tet-NICD mouse model, the cochleae were treated with 4-hydroxytamoxifen (20 ng / ml), Dox (Sigma, final concentration 2 μg / ml), LiCl (8 mM), forskolin (FSK) (20 μM), siFIR (Puf60, gene ID: 67959) (0.02 μM) and siMxi1 (gene ID: 17859) (0.02 μM); in the Atoh1-GFP mouse model, the cochleae were treated with VPA (2 mM), LiCl (8 mM), FSK (20 μM), siFIR (0.02 μM) and siMxi1 (0.02 μM) for 4 days and then treated with ad-Atoh1 (6 × 10 10pfu / ml) infection followed. siRNA was delivered according to the manufacturer's instructions (Polyplus-transfection; 89129-920). In cultured adult mouse cochleae, they were treated with HDAC inhibitors, belinostat (1 μM, Medchemexpress), trichostatin A (TSA) (82.5 nM, Medchemexpress), vorinostat (SAHA) (2 μM; Medchemexpress) and Myc siRNA Fbxw7 (0.02 μM, Santa Cruz Biotechnology). Controls were cultured adult cochleae of the same genotype treated with ad-Atoh1 in addition to vehicle (sterile water containing 0.1% DMSO). Cultures were placed in fresh medium for an additional 10–14 days. Cochleae were harvested and decalcified before immunohistochemistry.

[0094] Whole utricles were dissected from 4-week-old mice and cultured free-floating. Otoliths were removed using a gentle stream of phosphate-buffered saline (PBS) extruded from a 25G needle and syringe. Utricles were cultured in 1000 μL of medium in untreated 24-well flat-bottom plates. All cultures were maintained at 37°C, 5% CO2 / 95% air.

[0095] Culture medium consisted of Dulbecco's Modified Eagle Medium / F12 (DMEM / F12, Invitrogen) supplemented with B27 and N2. Neomycin sulfate stock solution (10 mg / mL in 0.9% NaCl, from Sigma Aldrich) was diluted to 4 mM in culture medium. Valproic acid (VPA) from Sigma was included at 2 mM and siRNA from Santa Cruz was included at 0.02 μM.

[0096] Genealogy Tracing Cochlear tissue from 4- to 6-week-old Sox2-CreER / tdT, Sox2-CreER / tdT / rtTA / tet-MYC quad, Sox2-CreER / tdT / rtTA / tet-NICD quad, and Sox2-CreER / tdT / rtTA / tet-MYC / tet-NICD quint mice was dissected for culture. 4-Hydroxytamoxifen (20 ng / mL) was added to the cultures on day 0 to activate Cre for lineage tracing studies. Ad-Atoh1-V5 virus was inoculated at 6x10 for 16 to 24 hours. 10 They were added to the medium at a concentration of pfu / ml.

[0097] Transtympanic injection of chemicals and siRNA in vivo All adult mice used were 4 to 6 weeks old. Transtympanic injections were performed 7 days after subcutaneous injection of kanamycin (0.8 mg / g; Sigma) 30 min followed by intraperitoneal injection of furosemide (0.3 mg / g; Hospira Inc). Mice were anesthetized by intraperitoneal injection of xylazine (10 mg / kg) and ketamine (100 mg / kg). Transtympanic injections were performed with 5 μl of chemical combinations of VPA (5 mg / ml), LiCl (40 mM), FSK (50 μg / ml), siFIR (0.6 μg / 10 μl) and siMxi1 (0.6 μg / 10 μl) or vehicle (ddH2O containing 0.5% DMSO). Mice were injected with chemicals or vehicle through the tympanic membrane (TM) in one ear. Microforceps were used to retract the skin to reveal the medial superior fold adjacent to the TM. A Hamilton syringe with a 33G needle was used to inject drugs transtympanically.

[0098] Virus injection in vivo All surgical procedures were performed in a clean, dedicated area. The equipment was thoroughly cleaned with 70% ethanol and autoclaved prior to surgery. Mice were anesthetized by intraperitoneal injection of xylazine (10 mg / kg) and ketamine (100 mg / kg). For virus injection, a cochleostomy was performed in anesthetized mice by opening the bulla, and 5 × 10 12 Adenovirus with a titer of pfu / ml was injected into the middle turn of the scala media with a pressure-controlled motorized microinjector at a rate of 3 nl / sec. A total of 1 μl of adenovirus was injected into each cochlea.

[0099] [Example 1] A novel combination of siRNA and chemical compounds for hair cell regeneration Various combinations of small molecules and siRNAs were screened for hair cell reprogramming and regeneration in cultured adult mouse cochleae. Small molecules and siRNAs included in the screen targeted various pathways including Notch, Myc, mTOR, Wnt, Tgfb, FGF, retinoic acid, BMP4 and Alk5 pathways. Methyltransferase inhibitors and HDAC inhibitors were also screened for their ability to reprogram and regenerate hair cells in cultured adult mouse cochleae. Among the various combinations tested, the combination of valproic acid (VPA) and two siRNAs (siFIR (siF) and siMxi1 (siM)) enhanced cochlear reprogramming. As shown in Figure 1A-1C, novel MYO7A+ / Atoh1-mCherry+HCs were detected after reprogramming with VPA and siF / M combination followed by addition of ad-Atoh1-mCherry in adult wild-type mouse cochleae in vitro. Other combinations failed to achieve hair cell regeneration (Figures 2A-2C).

[0100] Next, we tested whether treatment with the novel "VLFsiFsiM cocktail" could regenerate HCs in the utricle. After treatment with neomycin for 5 days, the cut utricles had a reduced total number of utricle HCs compared to untreated cut utricles (Figures 3A-3B). After treatment with neomycin, cultured adult utricles were treated with VPA / siF / siM cocktail for 14 days. Treated utricles showed an increased number of hair cells (MYO7A+ / Atoh1-GFP+) compared to the vehicle (DMSO)-treated group (Figures 3C-3D). Note that in the utricle, hair cells could also be regenerated by the "VLFsiFsiM cocktail" alone, which does not contain Atoh1.

[0101] Together, we demonstrated that treatment with the novel "VLFsiFsiM cocktail" was sufficient to reprogram the adult mouse cochlea and utricle for HC regeneration with or without Atoh1.

[0102] [Example 2] Hair cell regeneration in wild-type mice To investigate whether the Wnt and cAMP pathways are sufficient to reprogram the adult cochlea, we combined the above chemicals VPA, siFIR, and siMxi1 (VsiFsiM) with a Wnt agonist (LiCl, L) and the cAMP agonist forskolin (FSK, F) to create a "cocktail" containing five chemicals (VLFsiFsiM). Treatment with the cocktail caused adult wild-type mouse cochleae to re-express inner ear progenitor genes including Six1, Eya1, and Gata3, but not embryonic stem cell genes such as Nanog and Fut4 (Figures 4A-4B).

[0103] To further trace the source of HC regeneration potential, we added the VLFsiFsiM cocktail to reprogram adult Atoh1-GFP mouse cochleae, in which the Atoh1 enhancer drives expression of a nuclear GFP reporter in the Atoh1 lineage (Figure 5A). Following ad.Atoh1 induction, we found numerous Atoh1-GFP+ / MYO7A+HCs regenerated in the adult cochleae in vitro (Figures 5B-5G). In contrast, virtually no Atoh1-GFP+ / MYO7A+HCs were regenerated in control cochleae without cocktail treatment (Figures 5B-5G). We performed FM1-43 uptake experiments to study whether new HCs possess functional transduction channels. This fluorescent dye passes through functional transduction channels and is captured by HCs by their charge. Notably, FM1-43+ / Atoh1-GFP+ / MYO7A+ triple positive HCs were generated in the cocktail-treated group, indicating dye uptake by HCs (Figure 5H). Interestingly, we found Atoh1-GFP+ / MYO7A+ globular structures in the cocktail-treated group (Figure 5I), indicating that progenitor / stem cell signals may have been activated by reprogramming with the cocktail. We performed lineage tracing studies using Sox2CreER-tdTomato (tdT) mice, in which tamoxifen (TAM) treatment persistently labeled supporting cells with tdT. After cocktail treatment and ad-Atoh1 infection, newly regenerated hair cells (MYO7A+) were found to be tdT positive, demonstrating that they were derived from transdifferentiation of SOX2+ supporting cells (Figure 5J-5L).

[0104] Next, we tested whether HDAC inhibitors other than VPA could be used to reprogram adult wild-type cochleae for hair cell regeneration. The experiments described above were performed using the HDAC inhibitors TSA (82.5 nM), SAHA (2 μM) or belinostat (1 μM) in the cocktail instead of VPA. Interestingly, each HDAC inhibitor was sufficient to induce hair cell reprogramming and efficient regeneration in adult wild-type cochleae after Ad-Atoh1 infection (Figure 6).

[0105] To investigate whether siRNA targeting the Myc modulator Fbxw7 could be used to promote hair cell regeneration, adult wild-type mouse cochleae were treated with siRNA targeting Fbxw7 (siFbxw7) alone or in combination with VPA. Experiments were also performed using siFbxw7 in combination with siF and siM (Figure 7A). In cultured adult WT cochleae, VPA treatment and Ad-Atoh1 infection did not result in HCs (MYO7A) (Figure 7B). Fbxw7 siRNA treatment alone and Ad-Atoh1 infection did not regenerate hair cells (MYO7A+) (Figure 7B). In contrast, treatment with the HDAC inhibitors VPA and siFbxw7 alone or in combination with siF and siM followed by Ad-Atoh1 infection induced HC (MYO7A+) regeneration in cultured adult WT cochleae (Figure 7B). Quantification showed an increase in hair cells as a result of regeneration under the various tested conditions (Figure 7C).

[0106] Together, we demonstrated that treatment with the novel "VLFsiFsiM cocktail" was sufficient to reprogram adult mouse cochleae for HC regeneration with Atoh1. We also demonstrated that various HDAC inhibitors (VPA, TSA, SAHA or belinostat) can be used in combination with siRNA targeting Myc modulators Fir, Mix1 or Fbxw7 to regenerate HCs.

[0107] [Example 3] Robust regeneration of HCs in a severe HC-depleted mouse model To further explore our HC regeneration protocol for future clinical applications, we tested our protocol in a severe HC loss mouse model. We observed that more than 95% of OHCs disappeared 7 days after intraperitoneal (ip) injection of kanamycin and furosemide. Strikingly, almost all OHCs were wiped out in the apical-middle and mid-turn, while almost all SOX2+SCs were preserved, making this a suitable model for detecting SC-HC transdifferentiation in vivo (Figures 8A-8C). After severe loss of OHCs, we treated the damaged cochlea by injecting VLFsiFsiM cocktail, and then regenerated HCs by Ad.Atoh1mCherry infection via cochlear fenestration in the mid-turn of the cochlea. After 14–21 days, multiple Atoh1mCherry+ cells were detected throughout the SE region from the apex to the mid-turn of the cochlea, with the majority in the OHC region. In the OHC region, significantly more ESPN+ cells were observed throughout the cocktail-reprogrammed cochleae compared to vehicle-treated unreprogrammed cochleae (Figures 9A–9C and 10A–10B). Notably, a large number of newly regenerated HCs were SOX2+ / ESPN+HCs, reflecting their newly regenerated status (Figures 11A–11B). Consistent with that, the total number of non-transdifferentiated ESPN- / SOX2+SCs was significantly reduced in the reprogrammed cochleae, indicating transdifferentiation of SCs to HCs (Figure 11C). Robust regeneration of HCs in the OHC region was observed by video (data not shown).

[0108] Together, we demonstrated that treatment with the novel "VLFsiFsiM cocktail" in combination with Atoh1 was sufficient to reprogram the adult mouse cochlea and regenerate HCs in a severe HC reduction mouse model in vivo.

[0109] Other embodiments While the present invention has been described in conjunction with its detailed description, it is understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A composition for reprogramming an adult mammalian inner ear for hair cell regeneration comprising an effective amount of a histone deacetylase (HDAC) inhibitor and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or a combination thereof, comprising: contacting the inner ear of an adult mammal under conditions and for a time sufficient to produce a population of progenitor cells in the inner ear of said adult mammal; The composition.

2. The HDAC inhibitor may be sodium butyrate, trichostatin A, hydroxamic acid, cyclic tetrapeptide, trapoxin B, depsipeptide, benzamide, electrophilic ketone, fatty acid compound, pyroxamide, phenylbutyrate, valproic acid, hydroxamic acid, romidepsin, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589), entrepilistat (EGFR ...

2. The composition of claim 1, wherein the active ingredient is selected from the group consisting of entinostat (MS275), CI-994 (N-acetyldinaline, also tacedinaline), entinostat (SNDX-275; formerly MS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, gibinostat (ITF2357) and mocetinostat (MGCD0103).

3. 3. The composition of claim 2, wherein the HDAC inhibitor is valproic acid, trichostatin A, vorinostat (SAHA) or belinostat (PXD101).

4. The composition of any one of claims 1 to 3, wherein the one or more inhibitory nucleic acids are small interfering RNA (siRNA), small hairpin RNA (shRNA) or antisense oligonucleotides.

5. The composition of any one of claims 1 to 4, wherein the one or more inhibitory nucleic acids comprise inhibitory nucleic acids targeting Fir and Mxi1.

6. 6. The composition of claim 1, further comprising contacting the mammalian cochlea with a Wnt agonist and / or a cAMP agonist.

7. The composition of claim 6, wherein the Wnt activator is lithium chloride (LiCl) and / or the cAMP activator is forskolin.

8. 8. The composition of any one of claims 1 to 7, wherein the progenitor cells of the population express Six1, Eya1, Gata3, Sox2, Notch1, Hes5 or a combination thereof.

9. 9. The composition of any one of claims 1 to 8, wherein the contacting occurs in the inner ear of a subject.

10. 1. A composition for treating hearing loss or vestibular dysfunction in a subject, comprising: histone deacetylase (HDAC) inhibitors, and one or more inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, or combinations thereof; and Atoh1 activator The composition comprising:

11. The HDAC inhibitor may be sodium butyrate, trichostatin A, hydroxamic acid, cyclic tetrapeptide, trapoxin B, depsipeptide, benzamide, electrophilic ketone, fatty acid compound, pyroxamide, phenylbutyrate, valproic acid, hydroxamic acid, romidepsin, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589), entrepilistat (EGFR ...

11. The composition of claim 10, wherein the active ingredient is selected from the group consisting of entinostat (MS275), CI-994 (N-acetyldinaline, also tacedinaline), entinostat (SNDX-275; formerly MS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, gibinostat (ITF2357) and mocetinostat (MGCD0103).

12. 12. The composition of claim 11, wherein the HDAC inhibitor is valproic acid, trichostatin A, vorinostat (SAHA) or belinostat (PXD101).

13. 13. The composition of any one of claims 10 to 12, wherein the one or more inhibitory nucleic acids are small interfering RNA (siRNA), small hairpin RNA (shRNA) or antisense oligonucleotides.

14. The composition of any one of claims 10 to 13, wherein the one or more inhibitory nucleic acids target Fir and Mxi1.

15. The composition of any one of claims 10 to 14, wherein the Atoh1 activator is a nucleic acid encoding Atoh1.

16. The composition of claim 15 , wherein the nucleic acid encoding Atoh1 is contained in a vector.

17. The composition of claim 16 , wherein the vector is a viral vector.

18. 18. The composition of claim 17, wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, and a vaccinia viral vector.

19. 19. The composition of any one of claims 10 to 18, further comprising a Wnt agonist and / or a cAMP agonist.

20. 20. The composition of claim 19, wherein the Wnt activator is lithium chloride (LiCl) and / or the cAMP activator is forskolin.

21. 21. The composition of any one of claims 10 to 20, wherein the subject is a human patient having noise-induced permanent hearing loss, drug-induced hearing loss, age-related hearing loss, sudden sensorineural hearing loss, hearing loss due to a viral infection, tinnitus, vestibular dysfunction, or a combination thereof.

22. The composition of any one of claims 10 to 21, wherein the subject is a human.

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