CSF transport pathways for drug delivery to the inner ear

Abstract

The present disclosure relates to the delivery of various agents to the inner ear of a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 293,209, filed December 23, 2021. The contents of this application are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to the delivery of various agents to the inner ear of a subject. [Background technology]

[0003] Hearing loss affects approximately 12% of individuals over the age of 12, or approximately 30 million Americans (NIDCD 2010). The likelihood of having bilateral hearing loss doubles every decade after age 50, reaching a point where over 60% of people over age 70 have hearing loss. By 2050, an estimated 2.45 billion individuals are projected to have mild to complete hearing impairment. Current remedial therapies using hearing aids or cochlear implants remain insufficient to restore full capacity of hearing function. Gene therapy administered through intracochlear or intravestibular injections in early postnatal mice has recently been shown to restore hearing in models of genetic hearing loss (Bankoti et al., 2021). However, gene therapy beyond the postnatal stage has proven unsuccessful (Bankoti et al., 2021), and these procedures are invasive surgeries that can cause damage to inner ear structures. Treatments and methods to restore hearing loss are needed. Summary of the Invention

[0004] SUMMARY OF THE DISCLOSURE The present disclosure addresses the above-mentioned needs in several aspects.

[0005] In one aspect, the disclosure provides a method for delivering an agent to the inner ear of a subject, the method comprising administering the agent to cerebrospinal fluid (CSF) in the subject.

[0006] The agent can be a therapeutic agent. Examples of therapeutic agents include one or more selected from the group consisting of small molecules, large molecules, peptides, proteins, antibodies, nucleic acids, vectors, and cells. In one embodiment, the therapeutic agent is a nucleic acid or vector. The nucleic acid or vector can include a gene encoding a protein or polypeptide. Examples of genes, proteins, or polypeptides include VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN , DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2 , ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G. In one embodiment, the gene encodes one selected from the group consisting of SEQ ID NO: 1-15. In one embodiment, the gene is VGLUT3. In one embodiment, the gene encodes SEQ ID NO: 1.

[0007] In another embodiment, the therapeutic agent is a Jun N-terminal kinase inhibitor, a brain-derived neurotrophic factor ligand, a PPAR agonist, a gamma-secretase inhibitor, a beta-catenin stimulator, a stem cell stimulator, an activator of Lgr5 positive epithelial stem cell proliferation, a cell differentiation regulator, a sensory hair cell regeneration compound, a glutathione peroxidase stimulator, a vitamin K-dependent protein C stimulator, an otoprotectant, a chemoprotectant, a hair cell regeneration compound, a glycogen synthase kinase-3 inhibitor, a 5-HT 3 receptor antagonists, calcineurin inhibitors, free radical scavengers, anti-inflammatory agents, apoptosis inhibitors, neuroprotectants, cyclin dependent kinase-2 inhibitors, apoptotic protease activating factor 1 inhibitors, metabotropic glutamate receptor 7 antagonists, toll-like receptor (TLR) antagonists, TLR-2 antagonists, TLR-4 antagonists, TLR-9 antagonists, tropomyosin receptor kinase (Trk)-C agonists, heat shock protein stimulators, guanylate cyclase stimulators, PDE 5 inhibitors, antivirals, DNA polymerase inhibitors, transferase inhibitors, KCNC potassium channel 1 modulators, KCNC potassium channel 2 modulators, and HSF1 gene stimulators. For example, therapeutic agents include brimapitide, OTO-413, pioglitazone, OTO-510, NXT-596, FX-322, pioglitazone hydrochloride, PIPE-505, otopotin, LY-3056480, ebselen, SPI-3005, ancrod, sodium thiosulfate, ACOU-085, OTO-6XX, DB-020, ORC-13661, FX-345, alazasetron besylate, disfenton sodium, acetylcysteine, The compound may be selected from the group consisting of AC-102, AZD-5438, LPT-99, NT-12, OR-112, PGT-117, P-13, PIPE-336, OR-102C, ebselen, small heat shock proteins, TOP-M119, AP-001, ganciclovir, dendrogen B, AUT-00206, dexamethasone (DEX), DEX-salvianolic acid B (DEX-SAL) conjugate, HB-097, and Plexalis.

[0008] In further embodiments, the therapeutic agent is a cell, such as a stem cell. Examples of stem cells include embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells. The vector mentioned above can be a plasmid, a cosmid, an artificial chromosome, or a viral vector. The viral vector can be any suitable viral vector, including but not limited to adeno-associated viral (AAV) vector, adenoviral vector, and lentiviral vector. In a preferred embodiment, the viral vector is an AAV vector. The serotype of the AAV vector can be selected from the group consisting of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

[0009] The agents referred to above can be imaging agents. Examples of imaging agents include one or more selected from the group consisting of organic molecules, fluorophores, metal ions, salts or chelates, particles, peptides, proteins, nucleic acids, polymers, and liposomes.

[0010] In some embodiments, the methods described above can further include examining the level of the drug in the inner ear after the administering step.

[0011] In a second aspect, the disclosure provides a method of treating a subject having or at risk of developing hearing loss. The method comprises administering an effective amount of a therapeutic agent to the CSF in the subject. The therapeutic agent can be selected from the group consisting of a small molecule, a large molecule, a peptide, a protein, an antibody, a nucleic acid, a vector, and a cell.

[0012] In some embodiments, the therapeutic agent is a nucleic acid or vector. The nucleic acid or vector can include a gene encoding a protein or polypeptide. Examples of genes include VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59 , OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ES PN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G. In one embodiment, the gene encodes one selected from the group consisting of SEQ ID NO: 1-15. In one embodiment, the gene is VGLUT3. In one embodiment, the gene encodes SEQ ID NO: 1.

[0013] The agent can be (i) a nucleic acid that is an RNAi agent or an antisense sequence that comprises a sequence complementary to a segment of a target gene, or (ii) a vector that encodes an RNAi agent or an antisense sequence. Examples can include siRNA-based therapeutics, such as the HES gene regulator developed by Otologic Pharmaceutics Inc.

[0014] In some embodiments, the therapeutic agent is a Jun N-terminal kinase inhibitor, a brain-derived neurotrophic factor ligand, a PPAR agonist, a gamma-secretase inhibitor, a beta-catenin stimulator, a stem cell stimulator, an activator of Lgr5 positive epithelial stem cell proliferation, a cell differentiation regulator, a sensory hair cell regeneration compound, a glutathione peroxidase stimulator, a vitamin K-dependent protein C stimulator, an otoprotectant, a chemoprotectant, a hair cell regeneration compound, a glycogen synthase kinase-3 inhibitor, a 5-HT 3 receptor antagonists, calcineurin inhibitors, free radical scavengers, anti-inflammatory agents, apoptosis inhibitors, neuroprotectants, cyclin dependent kinase-2 inhibitors, apoptotic protease activating factor 1 inhibitors, metabotropic glutamate receptor 7 antagonists, toll-like receptor (TLR) antagonists, TLR-2 antagonists, TLR-4 antagonists, TLR-9 antagonists, tropomyosin receptor kinase (Trk)-C agonists, heat shock protein stimulators, guanylate cyclase stimulators, PDE 5 inhibitors, antivirals, DNA polymerase inhibitors, transferase inhibitors, KCNC potassium channel 1 modulators, KCNC potassium channel 2 modulators, and HSF1 gene stimulators. For example, therapeutic agents include brimapitide, OTO-413, pioglitazone, OTO-510, NXT-596, FX-322, pioglitazone hydrochloride, PIPE-505, otopotin, LY-3056480, ebselen, SPI-3005, ancrod, sodium thiosulfate, ACOU-085, OTO-6XX, DB-020, ORC-13661, FX-345, alazasetron besylate, disfenton sodium, acetylcysteine, The compound may be selected from the group consisting of AC-102, AZD-5438, LPT-99, NT-12, OR-112, PGT-117, P-13, PIPE-336, OR-102C, ebselen, small heat shock proteins, TOP-M119, AP-001, ganciclovir, dendrogen B, AUT-00206, dexamethasone (DEX), DEX-salvianolic acid B (DEX-SAL) conjugate, HB-097, and Plexalis.

[0015] In some embodiments, the therapeutic agent is a cell, such as a stem cell. Examples of stem cells include embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells.

[0016] The vector mentioned above can be a plasmid, a cosmid, an artificial chromosome, or a viral vector. The viral vector can be any suitable viral vector, including but not limited to adeno-associated viral (AAV) vector, adenoviral vector, and lentiviral vector. In a preferred embodiment, the viral vector is an AAV vector. The serotype of the AAV vector can be selected from the group consisting of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

[0017] The hearing loss referred to above can be a genetic hearing loss, such as an autosomal dominant hearing loss, an autosomal recessive hearing loss, or an X-linked hearing loss. The hearing loss can be an acquired hearing loss, such as a noise-induced hearing loss, an age-related hearing loss, a disease- or infection-related hearing loss, a head trauma-related hearing loss, or an ototoxic drug-induced hearing loss.

[0018] In a third aspect, the disclosure features a method for increasing the expression of a gene in inner ear cells of a subject, the method comprising administering a nucleic acid or vector comprising the gene to CSF ​​in the subject. Examples of inner ear cells include inner hair cells, outer hair cells, vestibular hair cells, cochlear cells, and vestibular supporting cells. The vector can be a plasmid, a cosmid, an artificial chromosome, or a viral vector (e.g., AAV vector, adenoviral vector, and lentiviral vector). In a preferred embodiment, the viral vector is an AAV vector. The serotype of the AAV vector can be selected from the group consisting of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. The subject can have or be at risk of developing hearing loss. The hearing loss can be genetic hearing loss (e.g., autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss) or acquired hearing loss (e.g., noise-induced hearing loss, age-related hearing loss, disease- or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss). In one embodiment, the method may further include assessing the subject's hearing prior to the administering step, after the administering step, or at both time points.

[0019] In the methods described above, the drug, therapeutic agent, nucleic acid, or vector is administered intracisternally or intrathecally.

[0020] The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will become apparent from the description and the claims. [Brief description of the drawings]

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] [Fig. 1A-1I] A series of graphs showing that cerebrospinal fluid tracer easily enters the cochlea via the cochlear aqueduct. Figure 1A shows the experimental setup for MRI and CT scans. Figure 1B shows the anatomical structure of the ear. Figure 1C shows dynamic contrast-enhanced magnetic resonance (DCE-MR) imaging after cisternal injection of Gadovist (0.6 kDa), showing the tracer entering the cistern and the cochlea (white arrows). Figures 1D and 1E are time series showing Gadovist in the subarachnoid space entering the inner ear through an opening identified as the cochlear aqueduct (red arrow in D). Figure 1F shows a time series of MRI regions of interest (ROI) mean (+ / - SEM), depicting Gadovist arriving outside the cochlear aqueduct, then traveling into the cochlear aqueduct, and finally arriving at the ear (arrival time statistics in Figure 4A). Figures 1G, 1H, and 1I show ROI-averaged (+ / - SEM) high spatial resolution CT time series performed after cisternal injection of Omnipaque (0.8 kDa) and confirm that the tracer signal increases initially in the aqueduct before migrating to the floor of the scala tympani (red arrow in G), and that no significant tracer reaches the scala vestibuli within 30 min of scanning. Repeated t-test against zero mean with Sidak's multiple comparison test; *P<0.0021. [Fig. 2A-2F]A series of graphs showing that the cochlear aqueduct is enveloped by a lymphoid membrane. Figure 2A is a schematic diagram of the cochlea. Figures 2B and 2C show that microspheres (0.2 μm) injected into the cisterna magna are present in the cochlear aqueduct and are often associated with fiber-like structures located within it. Microspheres are also present at the bottom of the scala tympani and in the cochlea. Figure 2D shows lymphatic marker analysis, revealing that the arachnoid membrane forms a diaphragm-like structure (green arrowhead) at the cochlear end of the aqueduct (red arrowhead) that is PROX1, CRABP2 and podoplanin positive but LYVE1 negative. There are Iba1 and LYVE1 positive macrophage-like cells (red arrowhead) attached to the diaphragm. Figure 2E shows that numerous microspheres injected into the cisterna magna are trapped in the membrane and phagocytosed by Iba1 positive microspheres. [Figures 3A-3I]3A and 3B show the auditory brainstem responses (FIG. 3A) and distortion components of otoacoustic emissions (FIG. 3B) of Slc17a8+ / + (green) and - / - (red) mice at baseline (dashed line, open circle) and 2 weeks post-injection (solid line, filled circle) treated with either aCSF or VGLUT3-AAV (rescued). Since injection of GFP-AAV did not affect hearing, aCSF and GFP-AAV were merged together and served as controls. Data are mean + / - SEM, and individual responses are shown in the background. ABR thresholds in VGLUT3-treated WTs were elevated by 25 and 16 dB at 32 and 40 kHz, respectively, when compared to baseline (two-way ANOVA, treatment factor: F(1,96) = 7.98, p = 0.0058; frequency factor: F(5,96) = 88.4, p < 0.0001, (A), while other frequencies remained unaffected. KO mice showed improved ABR thresholds at all frequencies except 48 kHz (two-way ANOVA, genotype factor: F(1,72) = 237, p < 0.0001; frequency factor: F(5,72) = 24.9, p < 0.0001). Hearing in KO mice was significantly improved compared to WTs, except at 24 kHz. The KO mice had thresholds 5 dB higher than the WT mice (two-way ANOVA, genotype factor: F(1,72) = 18, p < 0.0001; frequency factor: F(5,72) = 72, p < 0.0001; 24 kHz, p = 0.0065 by Sidak's post-hoc test). Outer hair cell function, as measured by distortion product emissions (B), was not affected by aCSF or viral treatment in either WT or KO mice. Figure 3C shows the average auditory brainstem response waveforms with SEM (shaded) for Slc17a8+ / + (green) and - / - (red) mice showing evoked responses with decreasing stimulus intensity.Figures 3D, 3E, and 3F show representative photomicrographs of Slc17a8+ / + (D) and - / - (E) mice treated with aCSF or rescued (F) stained at the basal, middle, and apical turns for CtBP2 (red), GluR2 (green), and myosin VIIa (blue), or VGLUT3 (white). Rows of inner hair cells (IHC) are marked. Bars are 5 µm. Figures 3G, 3H, and 3I show quantification of presynaptic ribbons (G), postsynaptic GluR2 (H), and paired synapses (I) from Slc17a8+ / + (green) and - / - (red) mice treated with aCSF (white bars) or rescued (black bars). Data are mean + / - SEM, individual values ​​are shown, N=3-10. Stars indicate significant differences; *: p<0.05; **: p<0.01; ***: p<0.0001; Three-way ANOVA with Sidak's post-hoc analysis. [Figure 4A-4G] A series of graphs showing that an Aqp4-independent but dispersive mechanism drives CSF transport to the inner ear. Figure 4A is MRI data showing that tracer arrives significantly faster in the cochlear aqueduct compared to the ear. Figure 4B shows time activity curves for all ROIs measured in CT. There is no significant uptake in the cochlear nerve. Figures 4C, 4D, and 4E show that there was no significant difference in tracer influx after CM injection in wild type and AQP4 knockout. Figure 4F shows tracer concentration in the cochlear aqueduct, shown in both time and space axes from CT images (dots) corresponding to the dispersion model prediction (solid line). Figure 4G shows the dispersion coefficient estimated from the data in the cochlear aqueduct, which resembles the Stokes-Einstein free diffusion of Omnipaque in water. [Diagram 5] It is shown that the microspheres are abundant in the cochlear aqueduct after 1 hour and reach the base of the ear after 24 hours. [Figure 6A-6C]Figure 6 shows that CSF-delivered virus results in significantly higher expression in hair cells compared to intravenously injected virus. Figure 6A shows a schematic of virus entering the ear from the CSF. Figure 6B shows the expression of virus in the inner ear after cisternal and intravenous injection. The virus is highly expressed in the bottom and middle of the cochlea of ​​animals injected into the cisterna magna. Figure 6C shows quantification of virus expression in the inner ear in cisternal injected animals, showing significantly higher expression in the bottom and middle of cisternal injected animals compared to intravenously injected animals. [Figure 7A-7B] Auditory brainstem response wave 1 amplitude (A) and latency (B) from aCSF-treated (dashed line, open circle) or rescued (full line, filled circle) Slc17a8+ / + (green) and - / - (red) mice show that the KO-rescued group recovered to 62% of the WT-treated group (two-way ANOVA, genotype factor: F(3,94) = 24, p < 0.0001; stimulus intensity factor: F(4,94) = 18.8, p < 0.0001). aCSF-treated mice Peak latencies of waves 1, 3, and 5 in treated (white bars) or rescued (black bars) Slc17a8+ / + (green) and - / - (red) mice. Data are mean + / - SEM, individual values ​​shown, N=6-10. Null symbols represent the absence of ABR responses for Slc17a8- / - untreated mice. Stars indicate significant differences; *: p<0.05; **: p<0.01; one-way ANOVA with Sidak's post-hoc analysis. [Figure 8] The location of the region of interest in CT is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure relates to the delivery of various agents to the inner ear of a subject that can be used to restore hearing loss. Certain aspects of the present disclosure are based, at least in part, on the unexpected discovery that hearing is rescued in adult deaf mice by using the cerebrospinal fluid pathway to deliver therapeutic agents, such as gene therapy agents, to the inner ear.

[0024] Cerebrospinal fluid as a delivery route to the inner ear Cerebrospinal fluid (CSF) flow through the glymphatic system has emerged as a novel approach for brain-wide delivery of drug delivery. The inner ear lymph and CSF are directly connected via the cochlear aqueduct, which exhibits lymphoid characteristics. Real-time magnetic resonance imaging, computed tomography, and optical fluorescence microscopy have shown that large CSF tracers reach the inner ear by dispersive transport via the cochlear aqueduct in adult mice.

[0025] As disclosed in the Examples herein, a single intracisternal injection of adeno-associated virus expressing VGLUT3 transduced inner hair cells, rescued hearing thresholds, and re-established auditory synapses in Slc17A8- / - mice, a model of autosomal dominant hearing loss. Thus, CSF transport comprises a novel route for effective gene delivery to the adult inner ear.

[0026] By 2050, an estimated 2.45 billion individuals are projected to have mild to complete hearing loss (Collaborators, 2021). Current remedial therapies using hearing aids or cochlear implants remain insufficient to restore full capacity of hearing function. Gene therapy administered through intracochlear or intravestibular injections in early postnatal mice has recently been shown to restore hearing in models of genetic hearing loss (Bankoti et al., 2021). However, gene therapy beyond the postnatal stage has proven unsuccessful (Bankoti et al., 2021), and these procedures are invasive surgeries that can cause damage to inner ear structures.

[0027] In the brain, cerebrospinal fluid (CSF) reaches deep brain regions by being transported along perivascular spaces in what is called the glymphatic system. Glymphatic fluid transport plays an important homeostatic role, as fluid outflow removes metabolic waste products such as amyloid-β, tau, and lactate (Iliff et al., 2012; Xie et al., 2013). Hair cells in the inner ear are protected by the blood-labyrinth barrier, but are also metabolically very active (Nyberg et al., 2019; Spinelli et al., 2012). However, the ear is similar to the brain and eye, which have few lymphatic vessels (Salt et al., 2015). Recently, a glymphatic clearance system has been described in the eye (Wang et al., 2020), raising the possibility that the ear, like other neural tissues, exports metabolic waste products by active CSF transport. From a therapeutic perspective, it is important to note that glymphatic fluid transport can bypass the blood-brain barrier (BBB) ​​and be used as a route to deliver BBB-impermeable drugs to the brain. Therapeutic agents in the CSF can reach deep brain structures within minutes by perivascular CSF transport (Lilius et al., 2019; Plog et al., 2018).

[0028] To the inventors' knowledge, there are no previous studies that have demonstrated the feasibility of using the CSF route to deliver gene therapy to rescue hearing in a mouse model of human hearing loss. Herein, the inventors evaluated the connection between the inner ear lymphatic fluid and the CSF surrounding the brain, and whether that connection could be used for drug delivery. It was found that CSF enters the inner ear within minutes, and this easily accessible CSF route can deliver a variety of agents, including viral gene therapy, to restore function of genetic mutations associated with hearing loss in an adult mouse model of human hearing loss. Thus, the CSF route can be used to deliver a variety of agents to the inner ear.

[0029] Therapeutic Agents As disclosed herein, the agent can be a therapeutic agent. Examples of therapeutic agents include a small molecule, a large molecule, a peptide, a protein, an antibody, a nucleic acid, a vector, or a cell.

[0030] In one embodiment, the therapeutic agent is a Jun N-terminal kinase inhibitor, a brain-derived neurotrophic factor ligand, a PPAR agonist, a gamma-secretase inhibitor, a beta-catenin stimulator, a stem cell stimulator, an activator of Lgr5 positive epithelial stem cell proliferation, a cell differentiation regulator, a sensory hair cell regeneration compound, a glutathione peroxidase stimulator, a vitamin K-dependent protein C stimulator, an otoprotectant, a chemoprotectant, a hair cell regeneration compound, a glycogen synthase kinase-3 inhibitor, a 5-HT 3 receptor antagonists, calcineurin inhibitors, free radical scavengers, anti-inflammatory agents, apoptosis inhibitors, neuroprotectants, cyclin dependent kinase-2 inhibitors, apoptotic protease activating factor 1 inhibitors, metabotropic glutamate receptor 7 antagonists, toll-like receptor (TLR) antagonists, TLR-2 antagonists, TLR-4 antagonists, TLR-9 antagonists, tropomyosin receptor kinase (Trk)-C agonists, heat shock protein stimulators, guanylate cyclase stimulators, PDE 5 inhibitors, antivirals, DNA polymerase inhibitors, transferase inhibitors, KCNC potassium channel 1 modulators, KCNC potassium channel 2 modulators, and HSF1 gene stimulators. For example, therapeutic agents include brimapitide, OTO-413, pioglitazone, OTO-510, NXT-596, FX-322, pioglitazone hydrochloride, PIPE-505, otopotin, LY-3056480, ebselen, SPI-3005, ancrod, sodium thiosulfate, ACOU-085, OTO-6XX, DB-020, ORC-13661, FX-345, alazasetron besylate, disfenton sodium, acetylcysteine, The compound may be selected from the group consisting of AC-102, AZD-5438, LPT-99, NT-12, OR-112, PGT-117, P-13, PIPE-336, OR-102C, ebselen, small heat shock proteins, TOP-M119, AP-001, ganciclovir, dendrogen B, AUT-00206, dexamethasone (DEX), DEX-salvianolic acid B (DEX-SAL) conjugate, HB-097, and Plexalis.

[0031] In another embodiment, the therapeutic agent is a compound that promotes proliferation and / or differentiation of inner ear cells, such as hair cells or supporting cells. Examples of such compounds include, but are not limited to, retinoid receptor signaling activators, Wnt signaling activators, bone morphogenetic protein (BMP) signaling inhibitors, cyclin-dependent kinase (CDK) activators, E-box-dependent transcription activators, Notch signaling activators, histone deacetylase (HDAC) inhibitors, protein degradation inhibitors, PI3K-Akt signaling inhibitors, and cAMP response element binding protein (CREB) activators. Additional examples are described in U.S. Patent Application Publication No. 2019 / 0010449, the disclosure of which is incorporated herein by reference.

[0032] In one example, the therapeutic agent comprises a fusosome. "Fusosome" refers to a bilayer of amphipathic lipids surrounding a lumen or cavity, and a fusogen that interacts with the amphipathic lipid bilayer. In some embodiments, the fusosome comprises a nucleic acid. In some embodiments, the fusosome is a membrane-encapsulated preparation. In some embodiments, the fusosome is derived from a source cell. The fusosome can take a variety of forms. For example, in some embodiments, the fusosomes described herein are derived from a source cell. The fusosomes can be or include, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, microparticles, or any combination thereof. In some embodiments, the fusosomes are naturally released from the source cell, and in some embodiments, the source cell is treated to enhance the formation of fusosomes. In some embodiments, the fusosomes are about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, the fusosomes include one or more synthetic lipids.

[0033] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. For example, in some embodiments, the fusosome bilayer of amphipathic lipids is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus, or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises one or more viral nonstructural proteins, e.g., in its cavity or lumen.

[0034] Fusosomes can have a variety of structures or properties that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid to create a particle that can fuse with a target cell. In embodiments, the nucleic acids encode proteins that have one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0035] Nucleic Acids and Vectors As disclosed herein, gene therapy can be delivered via recombinant expression cassettes or expression vectors. The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can replicate. The nucleic acid sequence can be "exogenous", meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell, but is in a location within the host cell nucleic acid where the sequence is not normally found. Examples of vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (such as YACs). A person skilled in the art will be well equipped to construct vectors through standard recombinant techniques.

[0036] The term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, such as miRNA, these sequences are not translated. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well, as described below.

[0037] Promoters and transgenes A promoter is a control sequence, a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control the transcription initiation and / or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0038] A promoter may be one that is naturally associated with a gene or sequence, such as may be obtained by isolating the 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are "non-naturally occurring", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression.

[0039] It may be important to use promoters and / or enhancers that effectively direct the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression. The promoters used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct the expression of the introduced DNA segment in vivo or in vitro (e.g., in large-scale production of recombinant proteins and / or peptides). The promoters may be heterologous or endogenous. The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those skilled in the art.

[0040] One aspect of the disclosure provides a polynucleotide comprising: (i) a regulatory sequence that is at least 85% (e.g., 90%, 95%, 96%, 96%, 98%, 99%, and 100%) identical to a promoter or enhancer sequence of a gene selected from the group consisting of SIX1, ATOH1, SOX21, MYO7a, those listed below, and those in Table A; and (ii) a transgene operably linked to the regulatory sequence, wherein the transgene is selected from the group consisting of VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB 3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, A The polypeptides are selected from the group consisting of RT1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G. The amino acid sequences and related nucleic acid sequences encoding these polypeptides are known in the art. Listed below in Table B are GenBank accession numbers for some examples.

[0041] In some embodiments, the transgene is selected from the group consisting of VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), and Cx31 (GJB3). Exemplary amino acid sequences are shown in Table C below.

[0042] Several genes are known to be specifically expressed in cochlear cells, such as hair cells (HC), supporting cells (SC), and spiral ganglion neurons (SGN). See table below. The methods described herein can also be used to increase expression of any of these genes in those cells. TIFF2025504263000002.tif77170

[0043] One or more genes are specifically expressed during the development of the cochlear sensory epithelium and are distinguished by their low or absent expression in the adult brain. See, e.g., U.S. Patent Application Publication Nos. 2021 / 0388045, 2020 / 0392516; Ahmed et al., Developmental Cell 22, 377-390, February 14, 2012; Wingard and Zhao, Frontiers in Cellular Neuroscience, volume 9, article 202, 2015; Lan et al., Gene Ther 27(7):329-337, 2020). All of these references are incorporated by reference in their entirety. The promoters of these genes allow for cochlear epithelial cell-selective gene expression. TIFF2025504263000003.tif250170TIFF2025504263000004.tif245170TIFF2025504263000005.tif250170TIFF20255042630 00006.tif250170TIFF2025504263000007.tif250170TIFF2025504263000008.tif249170TIFF2025504263000009.tif162170

[0044] In one embodiment, the transgene encodes the human vesicular glutamate transporter 3 (VGLUT3 gene) protein. An exemplary human VGLUT3 amino acid sequence (GenBank: AJ459241.1) (SEQ ID NO: 1) is shown in Table B. Listed below is its encoding nucleic acid sequence (Takamori et al., EMBO Rep. 3 (8), 798-803 (2002)). SEQ ID NO:16: TIFF2025504263000010.tif100170TIFF2025504263000011.tif156170

[0045] In one embodiment, the promoter or enhancer sequence is a MYO7a promoter or enhancer. MYO7a (or myosin 7A) promoter or enhancer refers to a regulatory polynucleotide sequence that comprises or consists of a sufficient nucleic acid sequence to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion, or vestibular ganglion, and has at least about 85% sequence identity to any of the following nucleotide sequences. Examples can also comprise or consist of any of the following sequences or functional fragments thereof. See, for example, US Patent Application Publication No. 2020 / 0392516. MYO7A promoter 1 (SEQ ID NO: 17): MYO7A promoter 2 (SEQ ID NO: 18): MYO7A promoter 3 (SEQ ID NO: 19): MYO7A promoter 4 (SEQ ID NO:20): TIFF2025504263000012.tif219170

[0046] In one embodiment, the promoter or enhancer sequence is a MYO6 promoter or enhancer. MYO6 (or myosin 6) promoter or enhancer refers to a regulatory polynucleotide sequence that comprises or consists of a sufficient nucleic acid sequence to direct expression of a downstream polynucleotide in an outer or inner hair cell, a vestibular hair cell, a spiral ganglion, or a vestibular ganglion, and has at least about 85% sequence identity to the following nucleotide sequence. Examples may comprise or consist of the following sequences or functional fragments thereof. See, for example, US Patent Application Publication No. 2020 / 0392516. MYO6 promoter (SEQ ID NO:25): TIFF2025504263000013.tif85170

[0047] In one embodiment, the promoter or enhancer sequence is an ATOH1 promoter or enhancer. By ATOH1 promoter or enhancer is meant a regulatory polynucleotide sequence that comprises or consists of a sufficient nucleic acid sequence to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion, or vestibular ganglion, and has at least about 85% sequence identity to the following nucleotide sequence. Examples may comprise or consist of the following sequences or functional fragments thereof. See, for example, US Patent Application Publication No. 2019 / 0010449. ATHO1 promoter 1 (SEQ ID NO:21): ATHO1 promoter 2 (SEQ ID NO:22): TIFF2025504263000014.tif73170

[0048] In one embodiment, the promoter or enhancer sequence is a SIX1 promoter or enhancer. A SIX1 promoter or enhancer refers to a regulatory polynucleotide sequence that comprises or consists of a nucleic acid sequence sufficient to direct expression of a downstream polynucleotide in an outer or inner hair cell, a vestibular hair cell, a spiral ganglion, or a vestibular ganglion, and has at least about 85% sequence identity to the following sequence or the nucleotide sequence of GH14J060640. Examples may include or consist of the corresponding sequence or a functional fragment thereof. SIX1 promoter (SEQ ID NO:23):

[0049] In one embodiment, the promoter or enhancer sequence is a SOX21 promoter or enhancer. A SOX21 promoter or enhancer refers to a regulatory polynucleotide sequence that comprises or consists of a sufficient nucleic acid sequence to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion, or vestibular ganglion, and has at least about 85% sequence identity to the following sequence or the nucleotide sequence of GeneHancer (GH) identifier GH13J094705. Examples may comprise or consist of the corresponding sequence or a functional fragment thereof. SOX21 promoter (SEQ ID NO:24):

[0050] Viral Vectors Any convenient virus can be used to deliver the vector of interest to the subject. Viruses of interest include, but are not limited to, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and lentivirus. Viral gene therapy vectors are well known in the art, see, for example, Heilbronn&Weger (2010) Handb Exp Pharmacal.197:143-70. Vectors of interest include integrating and non-integrating vectors, such as those based on retrovirus, adenovirus (AdV), adeno-associated virus (AAV), lentivirus, poxvirus, alphavirus, and herpesvirus.

[0051] In some cases, non-integrating viral vectors such as AAV can be utilized. In one aspect, non-integrating vectors do not cause any permanent genetic modification. The vector can target adult tissues to avoid subject being affected by constitutive expression from early developmental stages. In some cases, non-integrating vectors effectively incorporate safety mechanisms to avoid overgrowth of transgene-expressing cells. If cells start to grow rapidly, they may lose the vector (and the resulting protein expression).

[0052] Non-integrating vectors of interest include those based on adenoviruses (AdVs), such as gutless adenoviruses, adeno-associated viruses (AAVs), integrase-deficient lentiviruses, poxviruses, alphaviruses, and herpesviruses. In certain embodiments, the non-integrating vector used is an adeno-associated virus-based non-integrating vector similar to natural adeno-associated virus particles. Examples of non-integrating vectors based on adeno-associated virus include vectors based on any AAV serotype, i.e., AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAVII, and pseudotyped AAVs. Vectors of interest include those that can transduce a wide range of tissues with high efficiency, with low immunogenicity and excellent safety profile. In some cases, the vector can transduce postmitotic cells and maintain long-term gene expression (up to several years) in both small and large animal models of the relevant disorder.

[0053] Pharmaceutical Compositions The polynucleotides or vectors described herein can be incorporated into vehicles for administration to patients, such as human patients suffering from sensorineural hearing loss and / or vestibular dysfunction. Pharmaceutical compositions containing vectors, such as viral vectors, containing the polynucleotides described herein operably linked to therapeutic transgenes can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd Edition, Allen, L. Ed. (2013); incorporated herein by reference) and in a desired form, for example, in the form of a lyophilized formulation or an aqueous solution.

[0054] A mixture of a nucleic acid vector (e.g., a viral vector) containing a polynucleotide as described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (as described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation may be sterile and fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0055] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered if necessary, and liquid diluents may first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intracisternal, intrathecal, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perillin solution. An exemplary synthetic perillin solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl.sub.2), 1-10 mM glucose, and 2-50 mM HEPE, with a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, formulations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0056] Pharmaceutical compositions are typically formulated as liquids, semi-solids (e.g., gels) or solids (e.g., matrices, scaffolds, etc.) as needed. Liquid compositions can be formulated for administration by any acceptable route known in the art to achieve delivery of drugs, compounds, cells, or compositions to target tissues (e.g., the inner ear). Typically, these include injection or infusion into the CNS or PNS in a diffuse manner or targeted to the site of disease or pain, by administration routes including, but not limited to, intraocular, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and / or perispinal administration routes, by delivery via intracranial or intravertebral needles and / or catheters with or without pump devices. Upon injection or infusion, the drugs, compounds, cells, or compositions can migrate to the site of interest.

[0057] Treatment method In some embodiments, the present disclosure provides novel therapeutic strategies for treating hearing loss or balance loss associated with damage or loss of inner ear cells, e.g., cochlear hair cells or vestibular hair cells, respectively.

[0058] The compositions described herein may be administered to subjects with sensorineural hearing loss and / or vestibular dysfunction by a variety of routes, including local administration to the inner ear (e.g., intrathecal via the oval window, round window, or semicircular canal (e.g., horizontal canal), intraauricular via the oval window, round window, or semicircular canal (e.g., administration to the cochlea or vestibular hair cells), parenteral, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, irrigation, and oral administration. The most suitable route for administration in any given case will depend on the particular composition administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions may be administered once, or more than once (e.g., once per year, twice per year, three times per year, bimonthly, or monthly).

[0059] Subjects who may be treated as described herein are those who have or are at risk of developing sensorineural hearing loss and / or vestibular dysfunction (e.g., subjects who have or are at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects who have or are at risk of developing damage to cochlear hair cells (e.g., damage associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing damage to vestibular hair cells (e.g., damage associated with disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing sensorineural hearing loss, hearing loss, or auditory neuropathy, subjects who have or are at risk of developing vestibular dysfunction (e.g., dizziness, spatial disorientation, or disequilibrium), subjects who have tinnitus (e.g., tinnitus isolated or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects who have a genetic mutation associated with hearing loss and / or vestibular dysfunction, or subjects who have a family history of hereditary hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject has hearing loss and / or vestibular dysfunction associated with or resulting from loss of hair cells (e.g., cochlear or vestibular hair cells). The methods described herein may include screening the subject for mutations in genes known to be associated with hearing loss or vestibular dysfunction prior to treatment or administration of the compositions described herein. The subject can be screened for genetic mutations using standard methods known to those skilled in the art (e.g., genetic testing). The methods described herein may also include evaluating hearing and / or vestibular function in the subject prior to treatment or administration of the compositions described herein. Hearing can be assessed using standard tests such as hearing tests, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions.Vestibular function can be assessed using standard tests such as eye movement tests (e.g., electronystagmogram (ENG) or videonystagmogram (VNG)), stabilometry, rotation chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). The compositions and methods described herein may also be administered as a prophylactic treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, for example, patients with a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), patients with a genetic mutation associated with hearing loss or vestibular dysfunction who do not yet exhibit hearing loss or vestibular dysfunction, or patients who have been exposed to risk factors for acquired hearing loss (e.g., disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging).

[0060] The compositions and methods described herein can be used to promote or induce hair cell regeneration in a subject (e.g., cochlear and / or vestibular hair cell regeneration). Subjects that may benefit from compositions that promote or induce hair cell regeneration include those suffering from hearing loss or vestibular dysfunction as a result of hair cell loss (e.g., hair cell loss associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), as well as those with abnormal hair cells (e.g., hair cells that do not function properly compared to normal hair cells), damaged hair cells (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or reduced hair cell number due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., to increase survival of damaged hair cells, promote repair of damaged hair cells, or preserve hair cells in subjects at risk of hair cell loss (e.g., hair cell loss due to age, loud noise, disease or infection, head trauma, or exposure to ototoxic drugs)).

[0061] The compositions and methods described herein can also be used to prevent or reduce ototoxic drug-induced hair cell damage or death (e.g., cochlear and / or vestibular hair cell damage or death) in subjects who have been treated with ototoxic drugs, or who are currently undergoing or will soon begin treatment with ototoxic drugs. Ototoxic drugs are toxic to cells of the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., spatial disorientation, dizziness, or imbalance), tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic agents (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially at high doses), and quinines. In some embodiments, the methods described herein prevent or reduce hair cell damage or death associated with acoustic trauma, disease or infection, head trauma, or aging.

[0062] A transgene operably linked to a promoter or a functional portion or derivative thereof for treating a subject as described herein can be a transgene encoding a protein expressed in healthy hair cells (e.g., cochlear and / or vestibular hair cells, e.g., a protein that plays a role in hair cell development, function, cell fate specification, regeneration, survival, or maintenance, or a protein that is deficient in a subject with sensorineural hearing loss and / or vestibular dysfunction) or other therapeutic protein of interest. The transgene can be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss or vestibular dysfunction is associated with a particular genetic mutation, the transgene can be a wild type of the gene that is mutated in the subject, or if the subject has hearing loss associated with hair cell loss, the transgene can encode a protein that promotes hair cell regeneration), the severity of the subject's hearing loss or vestibular dysfunction, the health of the subject's hair cells, the subject's age, the subject's family history of hearing loss or vestibular dysfunction, or other factors.Proteins that may be expressed by a transgene operably linked to a promoter described herein for the treatment of a subject described herein include, but are not limited to, VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, and the like. , CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2 , EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G.

[0063] Treatment may include administering various unit doses of a composition containing a nucleic acid vector (e.g., an AAV viral vector) containing a promoter as described herein. Each unit dose typically contains a predetermined amount of the therapeutic composition. The amount administered, as well as the particular route of administration and formulation, is within the skill of those of ordinary skill in the clinical arts. The unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. Administration may be performed using a syringe pump to control the rate of infusion to minimize damage to the inner ear (e.g., the cochlea). If the nucleic acid vector is an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector). In this case, the viral vector is, for example, about 1 × 10 in a volume of 1 μL to 200 μL (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL). 10 Vector Genome (VG) ~ 1 × 10 15 It may be administered to the patient in a dose of VG.

[0064] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or spatial disorientation), reduce tinnitus, increase expression of a therapeutic protein encoded by a transgene, increase function of a therapeutic protein encoded by a transgene, prevent or reduce hair cell damage, prevent or reduce hair cell death (e.g., ototoxic drug-induced hair cell death, age-related hair cell death, or noise (e.g., acoustic trauma) associated hair cell death), promote or increase hair cell development, increase hair cell number (e.g., promote or induce hair cell regeneration), increase or promote hair cell survival, or improve hair cell function. Hearing may be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hearing measurements obtained before treatment. Vestibular function may be assessed using standard tests for balance and spatial disorientation (e.g., oculomotor testing (e.g., ENG or VNG), stabilometry, rotation chair testing, ECOG, VEMP, and specialized clinical balance testing) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to measurements obtained before treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech.The compositions described herein may also be administered in an amount sufficient to slow or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in subjects who carry a genetic mutation associated with hearing loss or vestibular dysfunction, who have a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), or who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, acoustic trauma, or infection) but who do not exhibit hearing loss or vestibular dysfunction (e.g., spatial disorientation, dizziness, or disequilibrium), or in subjects who exhibit mild to moderate hearing loss or vestibular dysfunction). In a nucleic acid vector administered to a subject, expression of a therapeutic protein encoded by a transgene operably linked to a promoter described herein may be assessed using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detection of protein or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to expression prior to administration of a composition described herein. The hair cell number, hair cell function, or function of the therapeutic protein encoded by the nucleic acid vector administered to the subject may be indirectly assessed based on hearing tests or vestibular function tests, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to the hair cell number, hair cell function, or function of the therapeutic protein before administration of a composition described herein. Hair cell damage or death can be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hair cell damage and death typically observed in untreated subjects.These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more after administration of the compositions described herein. The patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more after administration of the composition, depending on the dose and route of administration used for treatment. Depending on the results of the evaluation, the patient may receive additional treatment.

[0065] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. The compositions can include a polynucleotide described herein or a nucleic acid vector containing such a polynucleotide. The nucleic acid vector can be packaged in an AAV viral capsid (e.g., AAV1, AAV2, AAV2quad(YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B). The kit can further include a package insert instructing a user of the kit, such as a physician, to carry out the methods described herein. The kit can optionally include a syringe or other device for administering the composition.

[0066] definition The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer may contain entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids. The term "protein" as used herein refers to either dimers (i.e., two) or multimers (i.e., three or more) of polypeptides or single-chain polypeptides. Single-chain polypeptides of a protein may be linked by covalent bonds, e.g., disulfide bonds, or non-covalent interactions.

[0067] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing sugars, phosphates, and bases that are either purines or pyrimidines. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases that can be incorporated into a DNA or RNA polymer. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA, and RNA encoded by a gene.

[0068] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments are well known in the art (see, for example, Nelson, MAbs (2010) 2(1):77-83), and include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, and Fv; diabodies; linear antibodies; single-chain variable fragments (scFv), single-chain antibody molecules including, but not limited to, fusions of light and / or heavy chain antigen-binding domains with or without (and optionally in association with) a linker; and monospecific or multispecific antigen-binding molecules formed from antibody fragments (including, but not limited to, multispecific antibodies constructed from multiple variable domains lacking the Fc region).

[0069] As used herein, an "inhibitory nucleic acid" is a double-stranded RNA, RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a reduction in expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or its orthologue, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. Typically, expression of the target gene is reduced by 10%, 25%, 50%, 75%, or even 90-100%.

[0070] A "therapeutic RNA molecule" or "functional RNA molecule" as used herein can be an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that effects spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi) including siRNA, shRNA or miRNA that mediate gene silencing (see Sharp et al., (2000) Science 287:2431), and any other non-coding RNA such as "guide" RNA and CRISPR RNA (see Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248), and the like known in the art.

[0071] "Antisense" refers to a nucleic acid sequence that is complementary to the coding strand or mRNA of a nucleic acid sequence, regardless of length.Antisense RNA can be introduced into individual cells, tissues, or organoids.Antisense nucleic acid can contain modified backbones, such as phosphorothioates, phosphorodithioates, or other modified backbones known in the art, or can contain non-natural internucleoside linkages.

[0072] As referred to herein, a "complementary nucleic acid sequence" is a nucleic acid sequence that can hybridize with another nucleic acid sequence consisting of complementary nucleotide base pairs. "Hybridize" refers to pairing that forms a double-stranded molecule between complementary nucleotide bases under suitable stringent conditions (e.g., in DNA, adenine (A) base pairs with thymine (T) and guanine (G) base pairs with cytosine (C)). (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0073] As used herein, the term "siRNA" refers to a double-stranded RNA molecule that interferes with the expression of a particular gene or genes after transcription. In some embodiments, siRNA functions to interfere with or inhibit gene expression using the RNA interference pathway. Similar interference or inhibition effects can be achieved with one or more of short hairpin RNA (shRNA), microRNA (mRNA), and / or nucleic acids (such as siRNA, shRNA, or miRNA) that contain one or more modified nucleic acid residues, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), unlocked nucleic acid (UNA), or triazole-linked DNA. Optimally, siRNAs are 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and have a two-base overhang at their 3' end. These dsRNAs can be introduced into individual cells or culture systems. Such siRNAs are used to down-regulate mRNA levels or promoter activity.

[0074] An "imaging agent" is a compound that has one or more properties that allow its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeled moiety that allows detection. An imaging agent can be any chemical or substance used to provide a signal or contrast in imaging. Examples include organic molecules, metal ions, salts or chelates, particles, labeled peptides, proteins, polymers, or liposomes.

[0075] "Subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, the subject is a mammal, particularly a primate, especially a human. In some embodiments, the subject is a livestock animal, such as cows, sheep, goats, cattle, pigs, etc., poultry, such as chickens, ducks, geese, turkeys, etc., and pets, such as farm animals, especially dogs and cats. In some embodiments (e.g., in a research context), the subject mammal is, for example, a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig, such as an inbred pig. "Mammal" refers to any mammal, including, but not limited to, a human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow, or pig.

[0076] As used herein, the term "cochlear hair cells" refers to a group of specialized cells in the inner ear that are involved in sensing sound. There are two types of cochlear hair cells: inner hair cells and outer hair cells. Damage to cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are implicated in hearing loss and deafness.

[0077] "Supporting cells," as used herein with reference to the cochlear epithelium, include epithelial cells in the organ of Corti that are not hair cells, including inner pillar cells, outer pillar cells, inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and / or Claudius cells.

[0078] As used herein, the term "expression" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0079] As used herein, the term "hair cell specific expression" refers to the production of an RNA transcript or polypeptide primarily in hair cells (e.g., cochlear hair cells and / or vestibular hair cells) as compared to other cell types of the inner ear (e.g., spiral ganglion neurons, glia, or other inner ear cell types). Hair cell specific expression of a transgene can be confirmed by comparing transgene expression (e.g., RNA or protein expression) between various cell types of the inner ear (e.g., hair cells vs. non-hair cells) using any standard technique (e.g., quantitative RT PCR, immunohistochemistry, Western Blot analysis, or measuring the fluorescence of a reporter (e.g., GFP) operably linked to a promoter). A hair cell specific promoter directs at least 50% greater (e.g., 50%, 75%, 100%, 125%, 150%, 175%, 200% greater, or more) expression (e.g., RNA or protein expression) of a transgene to which it is operably linked in hair cells compared to at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) of the following inner ear cell types: border cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, inner sulcus cells, outer sulcus cells, spiral ridge cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, Schwann cells.

[0080] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or human cell, such as a human hair cell).

[0081] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or human cell, such as a human hair cell).

[0082] As used herein, the terms "increase" and "decrease" refer to modulation that results in an increase or decrease in the amount of a metric function, expression, or activity, respectively, compared to a reference. For example, after administration of a composition in a method described herein, the amount of a marker of a metric described herein (e.g., transgene expression) may increase or decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more in a subject compared to the amount of the marker before administration. Generally, the metric is measured after administration at a time when administration has had the recited effect, for example, at least 1 week, 1 month, 3 months, or 6 months after the treatment regimen has begun.

[0083] As used herein, "locally" or "local administration" refers to administration at a particular site in the body where a local, rather than a systemic, effect is intended. Examples of local administration are onto the skin of a subject, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional, lymph node, intratumor, to the inner ear, and to a mucous membrane, where administration is intended to have a local, not a systemic, effect.

[0084] As used herein, the term "operably linked" refers to a first molecule that can be linked to a second molecule, the molecules being positioned such that the first molecule affects the function of the second molecule. The term "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) that allows for the possibility that both components function normally and that at least one of the components can mediate a function that is extended to at least one of the other components. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, if the promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. In additional embodiments, two portions of a transcriptional regulatory element are operably linked to each other when they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. The two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without the presence of any intervening nucleotides.

[0085] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule to which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule that can transport another nucleic acid to which it is linked. Certain plasmids can autonomously replicate in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Certain plasmids can direct the expression of genes to which they are operably linked.

[0086] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids that control or help control gene transcription (e.g., polyadenylation signals). Examples of transcriptional regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0087] As used herein, the term "promoter" refers to the recognition site on DNA that is bound by RNA polymerase. The polymerase drives transcription of the transgene.

[0088] "Transgene" means a fragment of DNA that is artificially inserted into a cell and becomes part of the genome of the organism that develops from that cell. Such transgenes may contain genes that are partially or completely heterologous (i.e., foreign) to the transgenic organism, or may represent genes that are homologous to endogenous genes of the organism.

[0089] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those of the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to a given nucleic acid or amino acid sequence B, or to a given nucleic acid or amino acid sequence B (which may alternatively be stated as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to a given nucleic acid or amino acid sequence B, a given nucleic acid or amino acid sequence A having a particular percent sequence identity to a given nucleic acid or amino acid sequence B, or a given nucleic acid or amino acid sequence A having a particular percent sequence identity to a given nucleic acid or amino acid sequence B) may be expressed as follows, i.e. It is calculated as 100 multiplied by (fraction X / Y). where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It is understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B is not equal to the percent sequence identity of A to B.

[0090] The term "administer" or "administration" refers to a method of delivering an agent, compound, cell, or composition to a desired site of biological action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, intracolonic, rectal, or intraperitoneal delivery. The agent, compound, cell, or composition may be formulated for administration by any acceptable route known in the art to achieve delivery of drugs and biological agents to target tissues (e.g., the inner ear), including, but not limited to, parenteral, including oral, nasal, ocular, and intravenous. Specific routes of parenteral administration include, but are not limited to, intramuscular, subcutaneous, intraperitoneal, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal, and / or perispinal routes, via delivery via intracranial or intravertebral needles and / or catheters with or without pump devices. In one embodiment, the agent, compound, or composition described herein is administered intrathecally or intracisternally. In some embodiments, administration can be, for example, otic, intracochlear, intravestibular, or transtympanic administration by injection. In some embodiments, administration is directly into the inner ear, for example, injection through a round or oval, otic capsule, or vestibular canal. In some embodiments, administration is directly into the inner ear via a cochlear implant delivery system. In some embodiments, the substance is injected transtympanically into the middle ear. In certain embodiments, "causing to be administered" refers to administering a second component after a first component has already been administered (e.g., at a different time and / or by a different actor).

[0091] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the clinical pathological course. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, amelioration or alleviation of the condition, remission or improvement of prognosis. In some embodiments, the agents described herein are used to delay the onset of the disease or to slow the progression of the disease.

[0092] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0093] The phrase "pharmaceutical acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharma- ceutical acceptable carrier commonly used in the pharmaceutical industry.

[0094] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, cosmids, or artificial chromosomes, RNA vectors, viruses, or any other suitable replicon (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used for expression of transgenes as described herein include vectors that contain regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other useful vectors for expression of transgenes contain polynucleotide sequences that enhance the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for the selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0095] As used herein, the term "viral vector" or "viral vector" refers to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within the virion. Alternatively, in some contexts, the term "vector" may be used to refer only to the vector genome / vDNA.

[0096] As disclosed herein, several ranges of values ​​are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, is also specifically disclosed between the upper and lower limits of that range, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which either, either, or both limits are included within the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included herein.

[0097] The term "about" or "approximately" means within an acceptable range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Unless otherwise specified, the term "about" means within an acceptable range of error for a particular value.

[0098] "Mixture" is intended to include solutions, dispersions, suspensions, solid / liquid mixtures, and liquid / liquid mixtures. Solutions, unlike dispersions, suspensions, and mixtures, lack a discernible interface between their solubilizing molecules and the solvent. Thus, the term mixture can be used when a solid is in direct contact with a liquid (solution) and when the solid is merely carried or suspended by a liquid. In either case, the liquid can be referred to as the "solvent."

[0099] The term "fusogenic" describes the ability of a vesicle to fuse with and thus become part of a target cell membrane.

[0100] A "fusogen" is any substance that increases the ability of a lipid vesicle bilayer to fuse with and thus become part of a target cell membrane. Upon fusion, the lipid vesicle can release the contents of the vesicle into the interior of the cell. Fusogens can exclude stable vesicle formation and can destabilize the vesicle. EXAMPLES

[0101] Example 1 This example describes the materials and methods used in Examples 2-6 below.

[0102] animal We aged male C57BL / 6JRj mice (Janvier Labs, Le Genest Saint-Isle, France), Prox1-EGFP+ mice kindly provided by Dr. Kari Alitalo (Choi et al., 2011), on a C57BL / 6JRj background (Janvier Labs, Le Genest-Saint-Isle, France), by crossing Aqp4-knockout and VGLUT3-knockout mice (Vogl et al., 2016), kindly provided by Dr. Tina Pangrsic Vilfan, Department of Otolaryngology, University Medical Center, Gottingen, from 4 weeks to 3 months of age. Mice were primarily housed in groups (2–5 mice per cage) with a 12-h light / dark cycle (lights on at 6:00) and controlled temperature and humidity, fed regular rodent chow and provided sterilized tap water ad libitum. All experiments in Denmark were approved by the Animal Experiments Council under the Danish Ministry of Environment and Food (license numbers: 2015-15-0201-00535 and 2020-15-0201-00480). All procedures in Sweden followed the regulations of the Karolinska Institutet and were approved by the Regional Ethics Committee in Stockholm (Stockholm's Norra Djurforsoksetiska Namnd, 11778-2019). Procedures were performed in accordance with the European Directive 2010 / 63 / EU, taking great care to minimize the number of animals included in the studies.

[0103] Injection into the cisterna magna of mice The protocol used in this study to inject tracers and AAV into the cisterna magna (CM) is an adaptation from a previously described chronic cannula implantation technique (Xavier et al., 2018). Briefly, mice were anesthetized with a mixture of ketamine / xylazine (100 mg / kg, 10 mg / kg, respectively), and after the depth of anesthesia was confirmed by the cessation of reflexes, the incision area was properly shaved and sterilized using an ethanol 70% swab (Vitrex Medical A / S Herlev, DK), followed by an iodine solution (Povidone Iodine 7.5% Henry Schein, Melville, NY, USA) applied using a cotton swab. Before the start of surgery, analgesia was applied, lidocaine (0.05 ml, 0.2 mg / ml) was applied to the incision site, and buprenorphine (0.05 mg / kg) was injected subcutaneously for postoperative analgesia. After making a skin incision around the occipital crest, the neck muscles were separated to expose the CM below. A 30G needle (SOPIRA® Carpule 30G 0.3x12mm, Kulzer, Hanau, Germany) was attached to one end of a PE10 tube filled with ACSF, and the other end of the tube was connected to a syringe (Hamilton Syringe GASTIGHT®, 1700 series, 1710TLL, volume 100 μL, PTFE Luer lock, Reno, NV, USA) which was then placed in a syringe pump (LEGATO® 130 Syringe pump, KD Scientific, Holliston, MA, USA). A pump was used to aspirate the injection medium into the tube, and the needle was then punctured through the dura and inserted into the CM, held in place with a drop of cyanoacrylate adhesive (Loctite Super Glue Gel Control) cured with an adhesion promoter (Insta-Set™ CA Accelerator, Bob Smith Industries, Atascadero, CA, USA).

[0104] IV injection Four-week-old animals were placed under a heated ram and awake while 100 μL of a 1 / 10 diluted ssAAV-PHP.B / 2-hCMV-chI-EGFP-WPRE-SV40p(A) (4.7*10E13 vg / ml) solution was injected into the tail vein. After injection, animals were marked, placed in cages, and allowed to survive for 14 days before perfusion and tissues were processed as described below.

[0105] virus For eGFP virus injections, 10 μL of ssAAV-PHP.B / 2-hCMV-chI-EGFP-WPRE-SV40p(A) (4.7*10E13 vg / ml) (Viral Vector Facility VVF, Institute of Pharmacology and Toxicology, University of Zurich or Viral Core at Boston Children's Hospital, see below) was injected. For therapeutic viruses, AAV2 / Php.B-CBA-VGLUT3-WPRE (2.27E13) (Viral Core at Boston Children's Hospital, BCH) was used. AAV was injected at a controlled rate of 1 ul / min, and to minimize backflow, the needle and glue were removed only 30 min after the end of the injection. The skin was then sutured with 6-0 absorbable sutures (Vicryl, Ethicon) and upon awakening, the mice were administered carprofen (5 mg / kg, SC) and finally returned to their cages. Mice received carprofen daily until 72 hours after surgery. eGFP-injected mice were euthanized after a 2-week survival period and transcardially perfused as described below. Auditory brainstem responses of mice injected with therapeutic viruses were measured 2 weeks after injection.

[0106] Microspheres Mice were subjected to CM cannulation as described above. Prior to injection, amine-modified microspheres (Thermofisher) were diluted in ACSF at a ratio of 1:3 and subjected to ultrasonic vibration for 5 min. They were then injected at an injection rate of 1 ul / min and allowed to circulate for 1 and 24 h before the animals were perfused and tissues were processed as described below.

[0107] Histological and immunohistochemical examination perfusion Prox1-EGFP+, Slc17A8 mutant, and C57BL / 6JRj mice were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine (100 mg kg / 10 mg kg) and perfused transcardially with 10 mL 0.01 M phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich, St. Louis, Missouri, USA), followed by 30 mL of 4% paraformaldehyde solution (PFA, Sigma-Aldrich) diluted in PBS, pH adjusted to 7.4.

[0108] Virus and microsphere injected animals cryosectioning After perfusion, the ears of the microsphere-injected animals were harvested and placed in formaldehyde for 24 hours before storage in PBS at 5°C. The inner ears were decalcified in 0.12 M ethylenediaminetetraacetic acid (EDTA) for approximately 4 days before incubation in 20% sucrose in PBS overnight for dehydration. For cryosectioning, the cochleae were then embedded in gelatin for 3 hours at 37°C, after which they were incubated in 20% sucrose for 24 hours for dehydration. The cochleae were then frozen in tissue culture medium (SPECS) using dry ice and isopentane and sectioned in 10 μm thick sections.

[0109] GFP-treated cochleae were stained with mouse monoclonal GFP (1:400) for quantification of GFP-transfected inner hair cells (IHC) and with rabbit polyclonal carbainsin (1:400) or rabbit polyclonal myosin VIIA (1:400) for delineation of hair cell bodies. Cochlear samples were incubated overnight at 37°C with primary antibodies, followed by incubation for 2 h at 37°C with secondary antibodies conjugated to Alexa fluoride dyes, goat anti-mouse 488 (green, 1:1000) and goat anti-rabbit 594 or donkey anti-rabbit 594 (red, 1:1000), depending on the primary antibody. Cochlear sections were then mounted in Vectashield antifade mounting medium with DAPI (H-1200, Vector Laboratories), coverslipped, and sealed with nail polish.

[0110] Cochleae treated with VGLUT3 virus or aCSF (used as control) were stained overnight at 37°C for (a) C-terminal binding protein 2 (mouse IgG1 anti-CtBP2, 612044 from BD-Biosciences, used at 1:200) for demonstration and quantification of presynaptic ribbons, (b) glutamate receptor subunit A2 (mouse IgG2A anti-GluA2, MAB397 from Millipore, used at 1:800) for quantification of glutamatergic postsynaptic receptors, (c) anti-myosin VIIA (rabbit IgG, 25-6790 from Proteus Biosciences, used at 1:200) for delineation of hair cell bodies, and (d) anti-vesicular glutamate transporter 3 (guinea pig, anti-VGLUT3, AB5421-I from Sigma-Aldrich, used at 1:400) for observation of hair cell recovery. Incubation with secondary antibodies conjugated to Alexa fluorochromes was followed with (a) goat anti-mouse IgG1 647 (far red, 1:500), (b) goat anti-mouse IgG2a 555 (red, 1:500), (c) donkey anti-rabbit IgG 405 (blue, 1:200), and (d) goat anti-guinea pig IgG 488 (green, 1:500) corresponding to the primary antibodies for 2 h at 37° C. Cochlear slices were then mounted in Vectashield antifade mounting medium without DAPI (H-1000, Vector Laboratories), coverslipped, and sealed with nail polish.

[0111] Cochlear frequency mapping was then performed using a custom Image-J software plugin provided by the NIH (Measure_Line.class from the Liberman research group at the Eaton-Peabody Institute). This mapping provided the entire length of each mouse cochlea and several respective frequency points (apical, middle, basal) and was used as a guide to obtain high-quality images in a confocal microscope for discrete frequency regions across the entire length of the cochlea. Z-stacks (20-40) with dimensions 1024 × 1024 and 16-bit images were taken with a 63x oil immersion objective (NA 1.40) on a Zeiss LSM880 confocal microscope. Z-stacks with 642 nm and 1 μm spacing were used to capture all pre- and postsynaptic structures and their couplings of at least 10 IHCs.

[0112] Quantification of presynaptic and postsynaptic inner hair cell structure Surface and masking structures were created to assess the total number of ribbons and postsynaptic GluA2 puncta per IHC. Image stacks were analyzed using Imaris software (x64 9.2.0, Bitplane AG, Zurich, Switzerland) for the number of ribbons, postsynaptic receptors, and their synaptic pairings. Regions of interest (approximately 10 IHCs) were then tagged for counting by using the "spots" function. After adjusting the threshold, puncta with pixel intensity 0.7-0.8 μm on an 8-bit scale (0-255) were counted. "Distance transformation" and "spot colocalization" were used to determine synaptic pairings. The threshold / distance between spots was set to 1 μm.

[0113] Whole head for lymphatic marker analysis Whole mice and torsos of perfusion-fixed Prox1-EGFP+ mice were decalcified in 10% EDTA in Tris buffer (pH 7) at room temperature for 3 weeks before paraffin embedding and serial sectioning. Sections were processed for immunohistochemistry as described above.

[0114] Immunohistochemistry After PBS washes, tissue sections of ears, brains, and other analyzed organs were blocked in a PBS solution containing 0.3% TritonX-100 (Sigma-Aldrich) and 5% normal donkey or goat serum (Gibco™; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 1 h at room temperature (RT), followed by incubation overnight at 4°C with primary antibodies (Table S1) diluted in blocking solution. Immunolabeling was revealed by incubation with appropriate secondary antibodies conjugated to fluorophores (Alexa Fluor, 1:500; Invitrogen™ Molecular Probes™; Thermo Fisher Scientific) for 2 h at room temperature. DAPI (4',6-diamidino-2-phenylindole, Thermo Fisher Scientific, 1 g / mL diluted in PBS) was used for nuclear counterstaining, and sections were mounted on glass slides using Prolong Gold Antifade reagent (Invitrogen / Thermo Fisher Scientific, Carlsbad, California, USA). Images of immunolabeled sections and meninges were acquired with an epifluorescence microscope (Nikon Ni-E) equipped with a Plan Apo λ4X / 0.20 objective, and a confocal microscope (Nikon Eclipse Ti, Tokyo, Japan) equipped with Plan Fluor 20X / 0.75 and 40X / 1.30 oil objectives.

[0115] MR live imaging Immediately after the CM cannulation procedure, the animal was transferred to the MRI scanner and head movement during scanning was minimized by restraining the animal in an MR-compatible stereotactic holder with ear bars. Body temperature was maintained at 37 ± 0.5 °C using a thermostatically controlled water bed and monitored along with respiratory rate by an MR-compatible remote monitoring system (SA Instruments, NY, USA). MRI was performed on a 9.4T preclinical scanner (BioSpec 94 / 30 USR, Paravision 6.0.1 software, Bruker BioSpin, Ettlingen, Germany) equipped with a 1H cryo-cooled quadrature resonator Tx / Rx coil (CryoProbe, Bruker) and a 240 mT / m gradient coil (BGA-12S, Bruker). A modified scanning procedure was performed as previously described (Stanton et al., 2021). T2-weighted structural images were performed using 3D constructive interference steady-state (3D-CISS). All 3D-CISS images were calculated as maximum intensity projections from four realigned 3D-TrueFISP volumes with four quadrature encoding directions (TR / TE 3.9 / 1.95ms, Nex 2, FA 50°, FOV 19.2x12.8x12.8mm, Matrix 192x128x128, BW 150kHz). Flow-compensated 3D time-of-flight MR angiography (3D-TOF-MRA) was acquired with a FLASH sequence (TR / TE 10 / 1.95ms, Nex 2, FA 20°, FOV 19.2x15x16mm, Matrix 246x192x205, BW = 100kHz). For dynamic contrast-enhanced MRI (DCE-MRI), pre- and post-contrast T1-weighted images were acquired with a 3D-FISP sequence (TR / TE 4 / 2ms, FA 15°, FOV 19.2x12.8x12.8mm, Matrix 192x128x128, BW 150kHz).DCE-MRI of the whole mouse brain was performed for 1 or 0.5 min with an isotropic spatial resolution of 100 μm when the T1-enhancing contrast agent gadobutrol (20 mM; Gadovist, Bayer Pharma AG, Leverkusen, Germany) was injected into the cisterna magna (1 μL / min, 10 min). The time-series DCE scanning protocol included three baseline scans (3 min) followed by intracisternal injection. Scans lasted for 90–120 measurements (60–90 min).

[0116] CT live imaging During the CM cannulation procedure, a custom-made head bar was fixed to the top of the animal's skull. The head bar was designed to minimize undesired movements during image acquisition (Yoshida et al., 2016) and was fixed to the animal holder. Immediately after the CM cannulation procedure, the animal was moved to the CT scanner. The animal's breathing was monitored using Biovet software. CT scans were performed on a Vector4CT system (MILabs, Utrecht, Netherlands), and a scan of the whole mouse head was performed in 4.56 min with an isotropic spatial resolution of 20 μm. The time-series CT scanning protocol included one baseline scan (4.5 min) followed by intracisternal injection of Omnipaque (350 mgI / ml, 1 μL / min, 10 μL, GE healthcare). Scanning continued for six measurements (30 min) after intracisternal injection of Omnipaque.

[0117] Image Processing Both DCE-MRI and CT time series data were motion corrected using Advanced Normalization Tools (ANTs ver. 2.1.0) (Avants et al., 2008; Avants et al., 2011). The motion-corrected time series in MRI images were converted to percent change from baseline time series, calculated as the percent signal change from the mean signal of the baseline images. To correct for subject motion between MRI scans, the anatomical images (3D-CISS and 3D-TOF-MRA) were registered to the baseline images of DCE-MRI using two rigid registration processes in ITK-SNAP (ver. 3.8.0). In MRI, the 3D-CISS images were used as anatomical references to place volumes of interest (VOIs) within the cisterna magna, outside the cochlear aqueduct, within the cochlear aqueduct, and within the cochlea. The hyperintensities in 3D-CISS represent the CSF space and fluid inside the cochlea, therefore the VOIs mentioned above were semi-automatically segmented using thresholding based on the signal intensity of 3D-CISS using ITK-SNAP. VOIs were manually drawn outside the cochlear aqueduct based on the tracer accumulation and location of the cochlear aqueduct as revealed by maximum intensity projection of DCE-MRI images. The location of the CT VOIs is described below and the distances were measured manually in ITK-SNAP. An example of TIFF2025504263000015.tif248170 ROI location is shown in FIG.

[0118] CT Time-intensity data were extracted using ITK snap with the ROIs described above after background subtraction and then normalized to maximum cisternal intensity. Cochlear aqueduct dimensions were measured using the #X feature of ITK snap for #4 animals. Distances between ROIs were calculated using their spatial coordinates.

[0119] MR #3D image generation by Amira. Background subtracted images were normalized to maximum cisternal intensity. AQP4 statistical comparison (p-value) 3D Image Generation

[0120] Distributed Model We suggest that tracer transport through a narrow space such as the cochlear aqueduct or scala tympani of length L can be approximated by a dispersion equation: dC(x,t) / dt=D / nabla C(x,t), where x is the axial distance from the proximal end, t is time, and D is the (constant) dispersion coefficient. The bounds are given by Dirichlet conditions by making measurements at the proximal and distal positions. For the model of the cochlear aqueduct, C(0,t)=c_SAS(t),C(L,t)=c_coch.(t), where c_SAS and c_coch. are the tracer concentrations measured at the proximal and distal ends of the cochlear aqueduct. Similarly, for the scala tympani, C(0,t)=c_1(t),C(L,t)=c_#N(t), Here, the subscripts refer to the scala tympani VOI index.

[0121] This formula was derived from Fick's first law using a shell-based approach (Roselli, Diller 2011), assuming a flat concentration gradient at the channel boundary (we need to make concentration measurements from VOIs with diameters much smaller than their axial length).

[0122] To solve Eq. (#ref), we implemented a simple finite difference scheme in Python (#ref) using NumPy and Scipy (#ref). Estimation of the variance coefficients was achieved by minimizing the L2 norm prediction error using Scipy (#ref). E(D, data)=sum / _observations(prediction(x,t)-observation(x,t))^2

[0123] Viral vector production AAV9-PHP.B vectors carrying coding sequences for eGFP or mouse VGLUT3 were generated by the Viral Core at Boston Children's Hospital under the authority of the BCH Institutional Biosafety Committee (Protocol #IBC-P0000047). The AM / CBA-VGLUT3-WPRE-BGH plasmid (Akil et al., 2012), which contains mouse VGLUT3 cDNA under the control of the chicken β-actin (CBA) promoter and cytomegalovirus (CMV) enhancer, was kindly provided by Omar Akil. Plasmids were amplified, purified, and sequenced to confirm identity. Integrity of the ITRs was assessed by restriction digestion and then submitted to the Viral Core for packaging into AAV9-PHP.B capsids. Similarly, a plasmid containing eGFP driven by a CMV promoter was cloned into an AAV2 vector and packaged into AAV9-PHP.B capsids by Viral Core using a helper virus-free system and dual transfection method as previously described (Lee et al., 2020). The vector was purified via iodixanol step gradient ultracentrifugation followed by ion exchange chromatography. The titer of genome-containing particles of AAV9-PHP.B-CBA-VGLUT3-WPRE-BGH was 2.27×1013 gc / mL assessed by a Syber-Green-based qPCR assay using primers for the bovine growth hormone polyadenylation signal (bGH pA). Two independent batches of AAV9-PHP.B-CMV-eGFP-WPRE were used with titers of 3.54×1012 gc / mL and 4.49×1012 gc / mL, quantified using primers for GFP. Virus aliquots were kept at −80°C until use.

[0124] Auditory brainstem response and distortion product otoacoustic emissions Signal generation and acquisition were performed using Tucker-Davis Technologies System III hardware and software. Stimuli were generated using BioSigRZ software running on a PC connected to a signal processor (RZ6). For distortion products of otoacoustic emissions (DPOAE), two independently driven MF1 speakers combined into a custom-made one were used for closed-field stimulation. For auditory brainstem responses (ABR), stimuli were output through an open-field speaker (MF1). A Bruel and Kjaer 1 / 4-inch microphone and conditioning preamplifiers (4939 A 011 and 2690 A 0S1) were used to calibrate the stimulus levels. The speakers were calibrated one at a time using a frequency sweep (4–32 kHz). The output was corrected to produce a flat spectrum at 90 dB SPL (open-field speaker, ABR) and 80 dB SPL (closed-field speaker, DPOAE). Mice were anesthetized with a mixture of ketamine (Ketaminol 50 mg / ml, Intervet, 511485) and xylazine (Rompun 20 mg / ml, Bayer, KP0A43D) (100 and 10 mg / kg body weight, respectively) and placed in a custom-made acoustic enclosure with sound-absorbing material on the walls and ceiling. Body temperature was maintained at 36.5°C using a heating pad (Homeothermic Monitoring System 55-7020, Harvard Apparatus). The first DPOAE was recorded. Briefly, an acoustic coupler was inserted into the ear canal. A microphone (EK23103, Knowles) was inserted into the acoustic coupler connected to a preamplifier (ER-10B+, Etymotic Research) and a processor (200 kHz sampling rate) to measure the sound level in the ear canal. Each speaker reproduced one of two laryngeal source tones (f1 and f2), swept from 80 to 10 dB SPL (for f2) in 5 dB steps.The 2f1-f2 distortion components were measured at f2 = 8, 12, 16, 24, and 32 kHz, f2 / f1 = 1.25, and a stimulus level L1 = L2 + 10 dB SPL. ABRs were then measured. Stainless steel subdermal needle electrodes were placed at the vertex of the head (yang), below the right pinna (yin), and above the right leg (ground). ABRs were elicited by 8, 12, 16, 24, and 32 kHz tone bursts (0.5 ms rise / fall time, 5 ms duration) presented 21 times per second. Signals were collected via a low impedance headstage (RA4LI) connected to a preamplifier (RA4PA) and digitally sampled (200 kHz sampling rate). Responses to 200–3000 bursts were bandpass filtered at 0.3–3 kHz using BioSigRZ and averaged at each level. For each frequency, sound levels were decreased from 90 dB SPL in 5 dB steps. DPOAEs and ABRs were recorded 2-3 days before (baseline) and 14 days after (2 w) cisternal virus injection, at which point animals were euthanized for cochlear biopsy.

[0125] Example 2 CSF solutes enter the inner ear via the cochlear aqueduct To assess whether the inner ear is accessible to CSF ​​tracers in wild-type adult mice, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was utilized to visualize CSF transport of Gadovist (0.6 kDa). Real-time imaging showed that Gadovist (0.6 kDa) injected into the CSF-filled space of the cisterna magna (CM) reaches the cochlea and identified a CSF-filled channel connecting the subarachnoid space with the cochlea, which is consistent with the anatomical localization of the cochlear aqueduct (Figure 1A-D). Consistent with the cochlear aqueduct being the entry point, Gadovist signal rises first just outside the cochlear aqueduct, then at the cochlea, and finally at the cochlea (Figure 1D-F, S1A). The time to reach a maximum of 50 was significantly higher in the cochlea compared to all other groups (Figure 4A).

[0126] To define whether the cochlear nerve contributes to tracer entry into the cochlea, high spatial resolution computed tomography (CT) scans were performed. Omnipaque (0.8 kDa) was delivered into the cisterna magna, and serial CT scans confirmed that the tracer concentration increased, first reaching a maximum concentration in the initial segment of the cochlear aqueduct. The tracer then disperses further into the aqueduct before it reaches the bottom and central regions of the scala tympani (Figures 1G-I). No significant increase in tracer concentration was detected in the scala vestibuli or cochlear nerve during the 30 min scan (Figures 1I, 4B). Thus, the CT scans indicated that the aqueduct is the primary point of CSF tracer influx. To determine larger CSF tracer accessibility, animals were euthanized 1 and 24 h after CSF delivery of microspheres (0.2 μm). Histological analysis showed that microspheres were abundant in the cochlear aqueduct at both time points and were detected in the scala tympani after 24 h (Figures 2A and 5). Together, this analysis indicates that the cochlear aqueduct directly connects the floor of the scala tympani to the large CSF-filled subarachnoid space.

[0127] Example 4 An Aqp4-independent scattering mechanism drives CSF transport to the inner ear Since glymphatic fluid transport is facilitated by the water channel Aquaporin-4 (AQP4), it was next asked whether AQP4 plays a role in CSF influx into the inner ear. CSF influx into aqp4 knockout and wild-type littermates was compared after cisternal injection of Gadovist. Time-activity curves for Gadovist influx in mice lacking aqp4 did not differ from wild-type animals demonstrating that fluid flow is AQP4-independent (Figure 4A,C-D). Indeed, immunolabeling supported these observations by showing that cells lining the cochlear aqueduct were AQP4-negative (Figure 2F). To understand the mechanisms driving CSF transport into the inner ear, CT time-activity curves were used for the cochlear aqueduct and scala tympani as well as anatomical measurements to estimate effective diffusion constants. Deriving a diffusion-based model using Fick's second law corresponds to an estimated effective diffusion coefficient similar to pure diffusion for the tracer, which is possible because lymphoid membranes minimize the contributions of advection and diffusion, suggesting that the driving mechanism for CSF transport to the ear is aqp4-independent diffusion transport.

[0128] Example 3 The cochlear aqueduct exhibits lymphoid characteristics Because tracers easily enter the ear, it was hypothesized that the cochlear aqueduct is similar to Schlemm's canal, which drains aqueous humor from the anterior chamber of the eye (Aspelund et al., 2014). The narrow bony cochlear duct with a diameter of approximately 126 micrometers (Table 1) showed an adjacent thin dural layer covered by a very narrow claudin-11 positive arachnoid barrier cell layer. The duct was lined with a tubular eversion of the membrane that extended downwards until it terminated at its entrance to the cochlea, a distance of 439 ± 39 micrometers (Table 1). The membrane terminated as a diaphragm-like structure with an accumulation of macrophage-like cells on the cerebral side of the diaphragm. The membrane stained positive for CRABP2 (cellular retinoic acid binding protein 2) and lymphatic markers prospohomeobox protein 1 (Prox1) and podoplanin, but not VEGFR3 or lymphatic hyaluronan receptor (LYVE1) (Figure 2D). Prox1 is a master regulator of lymphatic endothelial cell fate, and lymphatic vessels expressing Prox1 have been shown to contribute to the drainage of macromolecules from the CSF (Louveau et al., 2018-2015 Nature paper). Schlemm's canal in the eye is similarly Prox-1 and podoplanin positive, but LYVE-1 and VEGFR3 negative (Aspelund et al., 2014). LYVE1 and Iba1 stained macrophages were associated with the membrane, but the cells forming the membrane were LYVE1 negative. Iba1 staining of microsphere images showed that Iba1 positive macrophages phagocytosed microspheres within the channel (Figure 2E).

[0129] Example 5 Viruses injected into the cisterna magna are expressed in the inner ear Can the cochlear aqueduct-CSF route be used as an alternative strategy for delivery of viral constructs to the inner ear? A reporter virus, AAV-PHP.B-CMV-EGFP, has previously been shown to induce robust expression in hair cells when injected directly into the ear (Lee et al., 2020). Adolescent mice received a single injection of AAV-PHP.B-CMV-EGFP and inner ears were harvested 14 days later. Inner hair cells were successfully transduced by the virus with a gradient of expression that was more robust at the base of the cochlea and decreased toward the apex in both the number of transfected cells and signal intensity (Figure 6A-B). This distribution is consistent with the hypothesis that the virus enters the inner ear through the aqueduct (Figure 6A). To control for BBB permeability, a subgroup of age-matched mice received AAV-PHP.B-CMV-EGFP intravenously, and the inner ear showed slight but evenly distributed viral expression when compared to CSF ​​delivery of the same construct (Figure 6A-B). In sum, the CSF can be used as a delivery route for viruses to the inner ear.

[0130] Example 6 CM delivery of VGLUT3-expressing virus rescues hearing in a mouse model of DFNA25 After determining the CSF pathway as an effective paradigm to virally transfect inner hair cells, we assessed whether virally mediated overexpression in the inner hair of Slc17a8, encoding vesicular glutamate transporter-3 (VGLUT3), could rescue autosomal dominant and progressive sensorineural hearing loss in a mouse model for 2-month-old adult Slc17a8- / - mice, DFNA25 (Ruel et al., 2008). VGLUT3 transports glutamate to synaptic vesicles before it is released into the synaptic cleft and is essential for transduction of signals from inner hair cells. Slc17a8- / - mice have hearing impairments due to defects in inner hair cells to take up and release glutamate to their afferent neurons, although the function of outer hair cells remains unaffected (Ruel et al., 2008). Auditory brainstem responses and distortion product otoacoustic emission baseline thresholds were measured in wild-type and Slc17a8- / - mice7 before either CSF or viral delivery of either EGFP- or VGLUT3-expressing AAVs, and 2 weeks post-injection (Figure 3). Mean auditory brainstem response baseline values ​​in WT ranged from 8 to 20 dB SPL at 8 to 24 kHz, whereas at 32 and 40 kHz, the mean values ​​were higher (between 38 and 74 dB SPL), a known effect seen in C57BL / 6J mice due to mutations in the age-related hearing loss gene (ahl gene) known to cause high-frequency hearing loss by 2 months of age (Johnson et al., 1997) (Figure 3A). In comparison, VGLUT3 KO mice had mean elevated baseline thresholds ranging from 74 to 95 dB SPL. DPOAEs were normal in both WT and KO mice (Figure 3B). Two weeks after administration of either vehicle (aCSF) or reporter gene (EGFP-AAV), no changes in hearing thresholds were found, confirming that intracisternal injections do not adversely affect hearing. In wild-type mice treated with VGLUT3-overexpressing AAV, thresholds increased by 25 and 16 dB at 32 and 40 kHz, respectively, when compared to baseline. In contrast, VGlut3 KO mice showed improved hearing thresholds at all frequencies except 48 kHz.In contrast, outer hair cell function, as measured by DPOAE, was not affected by aCSF or viral rescue in either WT or KO mice (FIG. 3B).

[0131] The average waveforms provide a visual impression of the degree of hearing rescue obtained in Slc17a8- / - mice after viral transfection of VGlut3 (Figure 3C). Overall, CSF delivery of AAV expressing VGLUT3 resulted in recovery of evoked responses in adult Slc17a8- / - mice, indicating that glutamatergic signaling in the inner hair follicles is restored. However, ABR wave 1 amplitude was restored to 62% of WT mice treated with VGLUT3-AAV, despite hearing thresholds reaching normal levels (Figure 3e). Wave 1 and 3 latencies were also found to be delayed by 0.2 ms (p=0.002) and 0.38 ms (p=0.0112), respectively, in rescued Slc17a8- / - compared to Slc17a8+ / + mice (Figure 3), whereas wave 5 latency was unaffected. These findings suggest that hearing thresholds were fully restored in Slc17a8− / − mice, but some degree of neural asynchrony remained 2 weeks after CSF delivery of VGLUT3-expressing AAV.

[0132] To determine the extent of synaptic repair by viral rescue, we quantified the number of paired synapses between inner hair cells and afferent dendrites using high-resolution immunocytochemistry for ribbons (CtBP2) and the AMPA subunit GluR2 in the apical, middle, and basal cochlear regions. Representative photomicrographs from different groups at each cochlear location are illustrated (Figure 3G). Viral rescue did not affect either the number of individual pre- and postsynaptic entities or their pairing in Slc17a8- / - mice. However, VGLUT3-AAV increased the number of ribbons and GluR2 units in the middle and basal turns, while synaptic pairing was fully restored in the basal region (for statistical analysis, see Supplementary Table 1, Figure 3).

[0133] Consideration The hearing organ is traditionally considered to be part of the peripheral nervous system. The inner ear together with the cochleovestibular ganglion originates from the surface ectoderm rather than the neuroectoderm that gives rise to the central nervous system (Freyer et al., 2011). However, the fluids of the ear and brain are connected via the cochlear aqueduct, and solutes are transported from the CSF-filled subarachnoid space to the inner ear within minutes. Here, we show that a large viral construct was successfully delivered through the cochlear aqueduct to the ear and restored hearing in deaf adult mice. Transport is driven by dispersion, in contrast to the advective Aqp4-dependent glymphatic transport in the brain. A pseudolymphatic membrane was identified in the cochlear aqueduct that appears to restrict transport between the ear and the brain, as evidenced by extensive phagocytosis of CSF microspheres by macrophages residing within the membrane. Nevertheless, reporter genes and VGlut3 were readily expressed by inner hair cells after intracisternal CSF administration. Successful delivery of viral gene therapy to treat hearing loss in the adult cochlea via the CSF has broad translational implications. Cisternal injection does not damage the cochlea in contrast to the traditional routes of direct injection through the round window membrane or the cochlea. Modern interventional radiology has made cisternal administration a relatively safe procedure, so CSF ​​delivery of gene therapy drugs will facilitate the treatment of genetically driven congenital hearing loss. While the present study evaluated hearing recovery 2 weeks after treatment, other studies have shown sustained AAV rescue of hearing up to 1 year after injection through the round window membrane (Gyorgy et al., 2019). In addition, intracisternal injections can be performed multiple times without risk of cochlear damage in contrast to round window injections. Overall, the present study provides proof of concept of a novel, relatively non-invasive and highly effective route for gene therapy of hearing disorders.

[0134] TIFF2025504263000016.tif246170

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[0136] The foregoing examples and descriptions of preferred embodiments should be understood as illustrating, rather than limiting, the present disclosure as defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the present disclosure as defined by the claims. Such variations are not to be considered as departing from the scope of the present disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for delivering an agent to the inner ear of a subject, comprising administering the agent to cerebrospinal fluid (CSF) in the subject.

2. The method of claim 1 , wherein the agent is a therapeutic agent.

3. 3. The method of claim 2, wherein the therapeutic agent is selected from the group consisting of a small molecule, a large molecule, a peptide, a protein, an antibody, a nucleic acid, a vector, and a cell.

4. The method of claim 3 , wherein the therapeutic agent is the nucleic acid or the vector.

5. The method of claim 4 , wherein the nucleic acid or vector comprises a gene encoding a protein or polypeptide.

6. The genes are VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, O TOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPR Q, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESP N,ESPNL,PRPH2,STRC,SLC8A2,ZCCHC12,LRTOMT2,LRTOMT1,USH1C,ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 6. The method of claim 5, wherein the polypeptide is selected from the group consisting of D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G.

7. The therapeutic agents include Jun N-terminal kinase inhibitors, brain-derived neurotrophic factor ligands, PPAR agonists, gamma-secretase inhibitors, beta-catenin stimulators, stem cell stimulators, activators of Lgr5 positive epithelial stem cell proliferation, cell differentiation regulators, sensory hair cell regeneration compounds, glutathione peroxidase stimulators, vitamin K-dependent protein C stimulators, otoprotectants, chemoprotectants, hair cell regeneration compounds, glycogen synthase kinase-3 inhibitors, 5-HT 3 receptor antagonists, calcineurin inhibitors, free radical scavengers, anti-inflammatory agents, apoptosis inhibitors, neuroprotective agents, cyclin dependent kinase-2 inhibitors, apoptotic protease activating factor 1 inhibitors, metabotropic glutamate receptor 7 antagonists, toll-like receptor (TLR) antagonists, TLR-2 antagonists, TLR-4 antagonists, TLR-9 antagonists, tropomyosin receptor kinase (Trk)-C agonists, heat shock protein stimulators, guanylate cyclase stimulators, PDE 5 inhibitors, antiviral agents, DNA polymerase inhibitors, transferase inhibitors, KCNC potassium channel 1 modulators, KCNC potassium channel 2 modulators, and HSF1 gene stimulators.

8. The therapeutic drug is brimapitide, OTO-413, pioglitazone, OTO-510, NXT-596, FX-322, pioglitazone hydrochloride, PIPE-505, otopotin, LY-3056480, ebselen, SPI-3005, ancrod, sodium thiosulfate, ACOU-085, OTO-6XX, DB-020, ORC-13661, FX-345, alazasetron besylate, disfenton sodium, acetylcysteine, AC-1 02, AZD-5438, LPT-99, NT-12, OR-112, PGT-117, P-13, PIPE-336, OR-102C, ebselen, small heat shock proteins, TOP-M119, AP-001, ganciclovir, dendrogen B, AUT-00206, dexamethasone (DEX), DEX-salvianolic acid B (DEX-SAL) conjugate, HB-097, and Plexalis.

9. The method of claim 3 , wherein the therapeutic agent is a cell.

10. The method of claim 9 , wherein the cell is a stem cell.

11. The method of claim 10, wherein the stem cells are embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells.

12. The method of claim 4 , wherein the vector is a plasmid, a cosmid, an artificial chromosome, or a viral vector.

13. The method of claim 12, wherein the viral vector is an AAV vector.

14. 14. The method of claim 13, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rhlO, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

15. The method of claim 1 , wherein the agent is an imaging agent.

16. 16. The method of claim 15, wherein the imaging agent is selected from the group consisting of an organic molecule, a fluorophore, a metal ion, a salt or chelate, a particle, a peptide, a protein, a nucleic acid, a polymer, or a liposome.

17. 17. The method of any one of claims 1 to 16, further comprising determining levels of the agent in the inner ear after the administering step.

18. 1. A method of treating a subject having or at risk of developing hearing loss, comprising administering to the CSF in said subject an effective amount of a therapeutic agent.

19. 20. The method of claim 18, wherein the therapeutic agent is selected from the group consisting of a small molecule, a large molecule, a peptide, a protein, an antibody, a nucleic acid, a vector, and a cell.

20. 20. The method of claim 19, wherein the therapeutic agent is the nucleic acid or the vector.

21. 21. The method of claim 20, wherein the nucleic acid or vector comprises a gene encoding a therapeutic protein or polypeptide.

22. The genes are VGLUT3, MYO7A (USH1B), USH1C, CDH23, PCDH15 (USH1F), SANS (USH1G), USH2A, ADGRV1 / VLGR1, WHRN (DFNB31), USH3A (CLRN1), HARS, Cx26 (GJB2), Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, O TOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPR Q, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESP N,ESPNL,PRPH2,STRC,SLC8A2,ZCCHC12,LRTOMT2,LRTOMT1,USH1C,ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 22. The method of claim 21, wherein the polypeptide is selected from the group consisting of D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, BIP, GJB2, and USH1G.

23. 23. The method of claim 22, wherein the gene is VGLUT3.

24. 21. The method of claim 20, wherein the agent is (i) the nucleic acid that is an RNAi agent or an antisense sequence that comprises a sequence complementary to a segment of a target gene, or (ii) the vector that encodes the RNAi agent or the antisense sequence.

25. The therapeutic agents include Jun N-terminal kinase inhibitors, brain-derived neurotrophic factor ligands, PPAR agonists, gamma-secretase inhibitors, beta-catenin stimulators, stem cell stimulators, activators of Lgr5 positive epithelial stem cell proliferation, cell differentiation regulators, sensory hair cell regeneration compounds, glutathione peroxidase stimulators, vitamin K-dependent protein C stimulators, otoprotectants, chemoprotectants, hair cell regeneration compounds, glycogen synthase kinase-3 inhibitors, 5-HT 19. The method of claim 18, wherein the therapeutic agent is selected from the group consisting of: adenosine triphosphate 3 receptor antagonists, calcineurin inhibitors, free radical scavengers, anti-inflammatory agents, apoptosis inhibitors, neuroprotective agents, cyclin dependent kinase-2 inhibitors, apoptotic protease activating factor 1 inhibitors, metabotropic glutamate receptor 7 antagonists, toll-like receptor (TLR) antagonists, TLR-2 antagonists, TLR-4 antagonists, TLR-9 antagonists, tropomyosin receptor kinase (Trk)-C agonists, heat shock protein stimulators, guanylate cyclase stimulators, PDE 5 inhibitors, antiviral agents, DNA polymerase inhibitors, transferase inhibitors, KCNC potassium channel 1 modulators, KCNC potassium channel 2 modulators, and HSF1 gene stimulators.

26. The therapeutic drug is brimapitide, OTO-413, pioglitazone, OTO-510, NXT-596, FX-322, pioglitazone hydrochloride, PIPE-505, otopotin, LY-3056480, ebselen, SPI-3005, ancrod, sodium thiosulfate, ACOU-085, OTO-6XX, DB-020, ORC-13661, FX-345, alazasetron besylate, disfenton sodium, acetylcysteine, AC-1 26. The method of claim 25, wherein the therapeutic agent is selected from the group consisting of AZD-02, AZD-5438, LPT-99, NT-12, OR-112, PGT-117, P-13, PIPE-336, OR-102C, ebselen, small heat shock proteins, TOP-M119, AP-001, ganciclovir, dendrogen B, AUT-00206, dexamethasone (DEX), DEX-salvianolic acid B (DEX-SAL) conjugate, HB-097, and Plexalis.

27. 20. The method of claim 19, wherein the therapeutic agent is a cell.

28. 28. The method of claim 27, wherein the cell is a stem cell.

29. 29. The method of claim 28, wherein the stem cells are embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells.

30. 21. The method of claim 20, wherein the vector is a plasmid, a cosmid, an artificial chromosome, or a viral vector.

31. 31. The method of claim 30, wherein the viral vector is an AAV vector.

32. 32. The method of claim 31 , wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rhlO, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

33. The method according to any one of claims 18 to 32, wherein the hearing loss is hereditary hearing loss.

34. 34. The method of claim 33, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

35. The method according to any one of claims 18 to 32, wherein the hearing loss is an acquired hearing loss.

36. 36. The method of claim 35, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

37. A method for increasing expression of a gene in inner ear cells of a subject, comprising administering a nucleic acid or vector containing the gene to CSF ​​in the subject.

38. 38. The method of claim 37, wherein the inner ear cell is selected from the group consisting of an inner hair cell, an outer hair cell, a vestibular hair cell, a cochlear cell, and a vestibular supporting cell.

39. 39. The method of claim 37 or 38, wherein the vector is a plasmid, a cosmid, an artificial chromosome, or a viral vector.

40. 40. The method of claim 39, wherein the viral vector is an AAV vector.

41. 41. The method of claim 40, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rhlO, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

42. 42. The method of any one of claims 37 to 41, wherein the subject has or is at risk of developing hearing loss.

43. 43. The method of claim 42, wherein the hearing loss is hereditary hearing loss.

44. 44. The method of claim 43, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

45. 43. The method of claim 42, wherein the hearing loss is an acquired hearing loss.

46. 46. ​​The method of claim 45, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

47. The method of any one of claims 18 to 46, further comprising assessing the hearing of the subject prior to the administering step.

48. 48. The method of any one of claims 18 to 47, wherein the method further comprises assessing the hearing of the subject after the administering step.

49. 49. The method of any one of claims 1 to 48, wherein the drug, therapeutic agent, nucleic acid, or vector is administered intracisternally or intrathecally.

50. The method according to claim 6 or 22, wherein the gene encodes one selected from the group consisting of SEQ ID NOs: 1 to 15.

51. 51. The method of any one of claims 6, 22, 23, and 50, wherein the gene encodes SEQ ID NO:1.

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