Cochlear cell-targeted gene therapy

Abstract

The present disclosure relates to methods for targeting specific cell types within the cochlea using optimized gene therapy vectors. Specifically, the present disclosure provides gene therapy vectors for specifically targeting cochlear cells and methods for treating hearing impairment and hearing loss-related disorders.

Description

[Technical Field]

[0001] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference in its entirety as a separate part of this disclosure and is identified hereinafter as: 54882_Seqlisting.txt, Size: 81,037 bytes, Created: October 19, 2020.

[0002] The present disclosure relates to methods for targeting specific cell types within the cochlea using optimized gene therapy vectors. Specifically, the present disclosure provides gene therapy vectors for specifically targeting cochlear cells and methods for treating hearing impairment and hearing-related disorders. [Background technology]

[0003] Hearing loss is a common sensory disorder worldwide, and much of prelingual hearing loss is due to genetic causes. Over 300 genetic loci associated with hereditary hearing loss and over 100 causative genes have been identified. Therefore, gene therapy is an attractive treatment for hearing loss. However, sensory cells in the adult mammalian cochlea lack the ability to self-renew, and invasive administration methods may increase damage to these cells.

[0004] The human inner ear is a small, three-dimensionally complex, fluid-filled structure encased in the body's densest bone and located deep at the base of the skull. Acoustic energy from sound is transmitted to the fluid in the cochlea via vibrations of the tympanic membrane and the ossicular chain of the middle ear, generating waves that travel along the basilar membrane. The length of the cochlea and the stiffness of the basilar membrane allow for the discrimination of audible frequencies. This in turn leads to the activation of mechanotransduction by hair cells, specialized sensory cells located in the organ of Corti, which transform mechanical stimuli into electrical depolarization. Electrical signals initiated by inner hair cells (IHCs) are then processed by spiral ganglion neurons (SGNs), which comprise the auditory nerve, and are ultimately decoded in the auditory cortex of the temporal lobe.

[0005] The organ of Corti contains two classes of sensory hair cells: inner hair cells (IHCs), which convert the mechanical information carried by sound into electrical signals transmitted to neural structures, and outer hair cells (OHCs), which amplify and modulate the cochlear response, a process necessary for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells at the edge of the spiral lamina, which are important for maintaining the adjacent tectorial membrane, and supporting cells, which have protective functions and can induce transdifferentiation into hair cells up to the early neonatal stage.

[0006] The inner ear is a fluid-filled space that is difficult to access. There are significant physical and diffusion barriers to accessing the inner hair cells, including the blood-labyrinth barrier, thus limiting systemic delivery of therapeutics to the inner ear. The blood-labyrinth barrier can slow the distribution of large molecules, such as viral vectors and other gene therapy reagents. Furthermore, disruption of the barrier can lead to leakage of high potassium into the perilymphatic space bathing the basolateral surface of hair cells, chronically depolarizing these cells and potentially leading to cell death. Furthermore, disruption of tight junctions between the endolymph and perilymph can lead to a decrease in the endocochlear potential, which reduces the driving force for sensory transduction in hair cells and therefore leads to decreased cochlear sensitivity and elevated hearing thresholds. (See Ahmed et al., J Assoc Res Otolaryngol. 18(5):649-670, 2017.)

[0007] It has been hypothesized that direct access to hair cells for gene therapy could be achieved by vector injection into the cochlear duct. However, direct injection into the cochlear duct alters the delicate, potassium-rich endolymphatic fluid within the duct, thereby disrupting the endocochlear potential and potentially causing sensory cell damage and irreversible hearing loss. The perilymph-filled spaces surrounding the cochlear duct, scala tympani, and scala vestibuli are accessible from the middle ear via the oval window membrane or round window membrane (RWM). The RWM, the only non-bony opening to the inner ear, is relatively easily accessible in many animal models, and administration of viral vectors using this route has been well tolerated (Askew et al., Sci Transl Med. 7(295):295ra108, 2015; Chien et al., Mol Ther. 24(1):17-25, 2016; Chien et al., Laryngoscope.;125(11):2557, 2016).

[0008] Viral gene therapy vectors, such as adenovirus, AAV, lentivirus, herpes simplex virus I, and vaccinia virus, have been tested in the cochlea, but these vectors only resulted in transient or suboptimal gene transfer (Fukui & Rapheal, Hear Res. 297:99-105, 2013). To date, only adenovirus has progressed into a clinical program (Luebke et al. Adv. Otorhinolaryngol:87-98, 2009). Previous studies of AAV serotypes for in vivo cochlear injection via different administration routes have highlighted the difficulty of targeting outer hair cells (OHCs), particularly via RWM injection (Liu et al., Mol Ther. 12(4):725-33, 2005), and have resulted in only partial rescue of hearing in mouse models of hereditary hearing loss (Akil et al., Neuron. 75(2):283-93, 2012; Askew et al., Science Trans. Med. 7:295ra108, 2015; Chien et al. Mol Ther. 24(1):17-25, 2016). The AAV vector Anc80L65 was shown to transduce OHCs with high efficiency when administered via injection into the round window membrane (Landegger et al., Biotechnology 35(3):280-284, 2017).

[0009] In particular, there are many challenges in targeting gene therapy vectors to inner hair cells in the cochlea. For example, there are very few inner hair cells in the cochlea. Furthermore, hair cells do not proliferate, and the final number of hair cells is reached early in development and does not increase in later years.

[0010] Currently, there is no biological treatment for hearing loss. Current state-of-the-art treatments focus on sound amplification and implanted electrodes that stimulate the auditory nerve. While these strategies provide partial restoration of function in limited patient populations, they do not approach restoration of natural hearing. Therefore, there is a need to develop methods for delivering gene therapy vectors to the relevant cell types of the organ of Corti within the cochlea that are not invasive or damaging to cochlear cells. [Prior art documents]

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Summary of the Invention

Means for Solving the Problems

[0012] The present disclosure provides gene therapy vectors that target specific cell types in the cochlea. These gene therapy vectors are useful for delivering transgenes to cells in the cochlea. The present disclosure provides a method for treating hearing loss, including administering the gene therapy vector using systemic delivery, such as intravenous delivery, intratympanic delivery, or intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. Gene therapy that targets specific cell types has advantages for treating hearing loss.

[0013] The term "hearing loss" is interchangeable with the term "hearing impairment," and these terms refer to hearing that is determined by a hearing test to be below the threshold level of normal hearing.

[0014] The present disclosure provides a method for delivering a transgene to cochlear cells of a subject, comprising administering to the subject a gene therapy vector encoding the transgene, wherein the gene therapy vector is administered to the subject using systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. For example, the disclosed method results in delivery of the transgene to cochlear cells, such as inner hair cells, outer hair cells, ganglion cells, supporting cells, Deiters cells, columnar cells, and / or epithelial cells.

[0015] The present disclosure also provides a method for treating hearing loss, such as hereditary hearing loss, age-related hearing loss, or hearing loss associated with injury or disease, comprising administering to a subject a gene therapy vector encoding a transgene, wherein the gene therapy vector is administered using systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. The provided method treats hearing loss caused by a genetic mutation or hearing loss caused by injury.

[0016] For example, the present disclosure provides methods of treating a subject suffering from a hearing loss-related disorder, such as Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejärg syndrome.

[0017] The present disclosure also provides a composition for delivering a transgene to cochlear cells of a subject, the composition comprising a gene therapy vector encoding the transgene, and the composition formulated for administration using systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. For example, the disclosed composition delivers the transgene to cochlear cells, such as inner hair cells, outer hair cells, ganglion cells, supporting cells, Deiters cells, columnar cells, and / or epithelial cells.

[0018] The present disclosure also provides a composition for treating hearing loss, such as hereditary hearing loss, age-related hearing loss, or hearing loss associated with injury or disease, wherein the composition comprises a gene therapy vector encoding a transgene, and the composition is formulated for systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. The provided composition is useful for treating hearing loss caused by a genetic mutation or hearing loss caused by injury.

[0019] For example, the present disclosure provides compositions for treating a subject suffering from a hearing loss-related disorder, such as Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejärg syndrome.

[0020] The present disclosure also provides the use of a gene therapy vector encoding a transgene for the preparation of a drug for delivering the transgene to cochlear cells of a subject, wherein the gene therapy encodes the transgene and the drug is formulated for systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to the cerebrospinal fluid. For example, use of the disclosed gene therapy vector results in delivery of the transgene to cochlear cells, such as inner hair cells, outer hair cells, ganglion cells, supporting cells, Deiters cells, columnar cells, and epithelial cells.

[0021] The present disclosure also provides a use of a gene therapy vector for preparing a medicament for treating hearing loss, such as hereditary hearing loss, age-related hearing loss, or hearing loss associated with injury or disease, comprising administering a gene therapy vector encoding a transgene to a subject, wherein the medicament is formulated for systemic delivery, such as intravenous (IV) delivery, intratympanic delivery, intrathecal delivery, or any other delivery method used to directly apply the vector to the cerebrospinal fluid. For example, the disclosed gene therapy vector can be used to prepare a medicament for treating hearing loss caused by a genetic mutation or hearing loss caused by injury.

[0022] For example, the present disclosure provides agents for treating a subject suffering from a hearing loss-related disorder, such as Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejärg syndrome.

[0023] Disclosed are methods, compositions, and uses for delivering any transgene of interest to cochlear cells, where the transgene is a polynucleotide sequence encoding a polypeptide of interest or a nucleic acid that inhibits, suppresses, or silences expression of the gene of interest, such as an siRNA or miRNA. Exemplary transgenes are polynucleotides encoding human atonal transcription factor (ATOH1) (Genbank Accession No. NM_005172.2; SEQ ID NO:2), otoferlin (SEQ ID NO:4), gap junction protein beta 2 (Genbank Accession No. NM_004004.6; SEQ ID NO:6), pendrin (SLC26A) (Genebank Accession No. XM_006716025.3; SEQ ID NO:8), forkhead box 1 (FOXG1) (Genebank Accession No. NM_005249; SEQ ID NO:10), activin A or inhibin (Genebank Accession No. NM_002192; SEQ ID NO:12), follistatin (FST) (SEQ ID NO:14), galectin-1 (Genbank Accession No. NM_002305.4; SEQ ID NO:19), or galectin-3 (Genbank Accession No. AB006780.1; SEQ ID NO:20). Additionally, the transgene is the wild-type nucleotide sequence of a gene listed in Table 1, Table 2, Table 3, Table 4 or Table 5 herein.

[0024] In any of the disclosed methods, compositions or uses, the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT or Anc80, AAV7m8, and derivatives thereof.

[0025] In any of the disclosed methods, the gene therapy vector comprises a CB promoter (SEQ ID NO: 1), a P546 promoter (SEQ ID NO: 16), a CMV promoter (SEQ ID NO: 17), or a Myo7A promoter (SEQ ID NO: 18).

[0026] Further, in any of the disclosed methods, compositions or uses, the gene therapy vector, composition or agent is administered using intrathecal delivery and the subject is placed in the Trendelenburg position after administration of the gene therapy vector, composition or agent. [Brief explanation of the drawings]

[0027] [Figure 1] 1 provides a schematic diagram of the AAV vectors tested in this disclosure. [Figure 2] Figure 1 shows delivery of the GFP transgene to the cochlea after intrathecal injection of the vector. Sagittal cryosections of the cochlea are stained with DAPI and GFP. Hematoxylin and eosin staining is provided for reference only. [Figure 3] DAPI and GFP staining in the cochleae of four different mice (shx2IT-LF, shx2IT-2F, shx2IT-LE, and shx2IT-LFLR) after intrathecal injection of AAV vectors is provided. [Figure 4] 1 provides DAPI and GFP staining in the mouse cochlea after intrathecal injection of AAV vectors, and different regions of the mouse cochlea are shown. [Figure 5] 1 provides DAPI, GFP, and actin staining in the mouse cochlea after intrathecal injection of AAV vectors. [Figure 6]Shown is DAPI and GFP staining in the mouse cochlea after intrathecal injection of DAPI and GFP. Cuts in this case were oriented in different directions to allow visualization of targeting throughout different regions of the cochlea. [Figure 7] DAPI and GFP staining in the mouse cochlea after intrathecal injection is shown, with cuts oriented in different directions to allow visualization of targeting throughout different regions of the cochlea. [Figure 8] Provides staining of DAPI, GFP, and actin in the mouse cochlea-vestibule after intrathecal injection. [Figure 9] Staining of DAPI, GFP, and actin (phalloidin) in the cochlea of ​​a mouse exposed to damaging noise and injected with AAV immediately after exposure to the noise is provided. Numbered sections are areas imaged at higher magnification. [Figure 10] We provide staining of DAPI, GFP, and actin (phalloidin) in the cochlea of ​​a mouse exposed to damaging noise and injected with AAV immediately after exposure to the noise. The section labeled 1 is the region of the cochlea imaged at higher magnification. [Figure 11] Staining of DAPI, GFP, and actin (phalloidin) in the cochlea of ​​a mouse exposed to damaging noise and injected with AAV immediately after exposure to the noise is provided. Numbered sections are areas imaged at higher magnification. [Figure 12] Staining of DAPI, GFP, and actin (phalloidin) in the cochlea of ​​a mouse exposed to damaging noise and injected with AAV immediately after exposure to the noise is provided. Numbered sections are areas imaged at higher magnification. [Figure 13] We provide staining of DAPI, GFP, and myo7A in the cochleae of noise-exposed mice and intrathecally injected AAV 24 h after noise exposure. [Figure 14]Staining of DAPI, GFP, and myo7A in the cochlea of ​​noise-exposed mice was performed 24 hours after noise exposure and intrathecal injection of AAV. Numbered sections are areas imaged at higher magnification. [Figure 15] We provide staining for DAPI, GFP, and myo7A in the cochleae of multiple noise-exposed mice and intrathecally injected AAV immediately (0 hpi) or 24 h (24 hpi) after noise exposure. [Figure 16] DAPI and GFP staining in the cochlea of ​​a noise-exposed mouse was shown, followed by an AAV injection immediately after noise exposure (0 hpi). This image shows an IT-injected mouse imaged at optimal exposure at 24 hpi. This image shows that GFP expression in the IT-injected animal at 24 hpi is lower than that in the IT-injected animal at 0 hpi, because imaging at optimal exposure at 24 hpi overexposes the 0 hpi image. [Figure 17] Hematoxylin and eosin staining of the cochlear nerve is provided. Abbreviations indicate the location of structures in the organ of Corti: SL = spiral lamina margin, TM = tectorial membrane, OHC = outer hair cell, IHC = inner hair cell, BM = basilar membrane. [Figure 18] 1 provides DAPI and GFP staining in the mouse cochlea after intravenous injection of AAV. [Figure 19] Shown are stainings of DAPI, GFP, and MyoD in cochleae from multiple mice after intravenous injection of AAV. Each panel is an image of a cochlea from a different mouse (top row is 10x magnification, bottom row is 20x magnification). [Figure 20] Provides DAPI and GFP staining in the mouse cochlea after intravenous injection of AAV without noise damage. [Figure 21] Provides DAPI and GFP staining in the mouse cochlea after intravenous injection of AAV without noise damage. [Figure 22] Provides DAPI and GFP staining in the mouse cochlea after intravenous injection of AAV without noise damage. [Figure 23]DAPI and GFP staining in the cochleae of control mice exposed to noise injury but not receiving intravenous injection of AAV is provided. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure provides an optimized AAV gene therapy for targeting the cochlea to treat hearing loss disorders. While the data provided herein focuses on the administration of AAV9 vectors, the present disclosure contemplates the use of any gene therapy vector containing a promoter that specifically targets cochlear cells, and these optimized vectors are administered using intravenous (IV) delivery, intrathecal delivery, or any other delivery method that accesses the cerebrospinal fluid (CSF). For example, data have shown that AAV9 injected directly into the cerebrospinal fluid via intrathecal injection is effective in targeting transgene expression in the inner hair cells of the cochlea. Therefore, intrathecal injection can be used to deliver gene therapy vectors to the cochlea, particularly to deliver gene therapy vectors to inner hair cells.

[0029] Gene Therapy Vectors Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, containing two 145-nucleotide inverted terminal repeats (ITRs). The term can refer to the virus itself or its derivatives. Unless otherwise specified, the term encompasses all subtypes, as well as both naturally occurring and recombinant forms. There are multiple serotypes of AAV. Each AAV serotype is associated with a specific clade, whose members share serological and functional similarities. Thus, AAVs may be referred to by clade. For example, AAV9 sequences are referred to as "clade F" sequences (Gao et al., J. Virol., 78:6381-6388 (2004)). The present disclosure contemplates the use of any sequence within a particular clade, such as clade F. The nucleotide sequences of the genomes of AAV serotypes are known.For example, the complete genome of AAV-1 is provided in GenBank accession number NC_002077, the complete genome of AAV-2 is provided in GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided in GenBank accession number NC_1829, the complete genome of AAV-4 is provided in GenBank accession number NC_001829, the AAV-5 genome is provided in GenBank accession number AF085716, the complete genome of AAV-6 is provided in GenBank accession number NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively, and the AAV-9 genome is described in Gao et al. The AAV-10 genome is provided in Mol. Ther., 13(1):67-76(2006), the AAV-11 genome is provided in Virology, 330(2):375-383(2004), a portion of the AAV-12 genome is provided in GenBank accession number DQ813647, and a portion of the AAV-13 genome is provided in GenBank accession number EU285562. The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. The sequence of the AAV-B1 genome is provided in Choudhury et al., Mol. Ther., 24(7):1247-1257(2016). Anc80 is an AAV vector for AAV1, AAV2, AAV8, and AAV9. The sequence of Anc80 is provided in Zinn et al., Cell Reports 12:1056-1068, 2015, Vandenberghe et al., PCT / US2014 / 060163, and GenBank accession numbers KT235804-KT235812, both of which are incorporated herein by reference in their entireties.

[0030] Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites contribute to the production of the three related capsid proteins. A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0031] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cell types, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The native AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. In some cases, the rep and cap proteins are provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0032] As used herein, the term "AAV" refers to wild-type AAV virus or virus particles. The terms "AAV," "AAV virus," and "AAV virus particle" are used interchangeably herein. The term "rAAV" refers to recombinant AAV virus or recombinant infectious encapsulated virus particles. The terms "rAAV," "rAAV virus," and "rAAV virus particle" are used interchangeably herein.

[0033] The term "rAAV genome" refers to a polynucleotide sequence derived from a native AAV genome that has been modified. In some embodiments, the rAAV genome has been modified to remove the native cap and rep genes. In some embodiments, the rAAV genome comprises endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises ITRs from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome comprises a transgene of interest flanked on the 5' and 3' ends by inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises a "gene cassette."

[0034] The term "scAAV" refers to a rAAV virus or rAAV viral particle that contains a self-complementary genome. The term "ssAAV" refers to a rAAV virus or rAAV viral particle that contains a single-stranded genome.

[0035] In some embodiments, the rAAV genome provided herein comprises one or more AAV ITRs flanking the transgene polynucleotide sequence. The transgene polynucleotide sequence is operably linked to transcriptional control elements (including, but not limited to, promoters, enhancers, and / or polyadenylation signal sequences) that are functional in the target cell to form a gene cassette. Examples of promoters are the CMV promoter, chicken β-actin promoter (CB), P546 promoter, and Myo7A promoter. Additional promoters are contemplated herein, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (such as, but not limited to, the actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, and creatine kinase promoter).

[0036] Further provided herein are CMV promoter sequences, CB promoter sequences, P546 promoter sequences, Myo7A promoter sequences, and promoter sequences that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a CMV (SEQ ID NO: 17), CB (SEQ ID NO: 1), Myo7A (SEQ ID NO: 18), or P546 (SEQ ID NO: 16) sequence that exhibit transactivation activity.

[0037] Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that allow specific expression in neurons or astrocytes. Examples include the neuron-specific enolase and glial fibrillary acidic protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline-regulated promoter. The gene cassette may also include an intron sequence to facilitate processing of the transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron.

[0038] "Packaging" refers to the series of intracellular events that result in the assembly and encapsidation of AAV particles. The term "production" refers to the process of producing rAAV (infectious, encapsulated rAAV particles) by packaging cells.

[0039] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins, respectively, of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0040] "Helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenovirus, herpesvirus, and poxvirus such as vaccinia. Adenoviruses can include many different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and can be obtained from depositories such as ATCC. Herpes virus includes, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which can also be obtained from depositories such as ATCC.

[0041] "Helper virus functions" refer to functions encoded in the helper virus genome that enable AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a number of ways, including by providing a helper virus or, for example, by providing polynucleotide sequences encoding the necessary functions to the producer cell in trans.

[0042] The rAAV genome provided herein lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome (e.g., ITR) contemplated herein can be from any AAV serotype suitable for deriving recombinant viruses, including, but not limited to, AAV serotypes Anc80, Anc80L65, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV7mb, AAV-8, AAV-9, AAV-10, AAV-RH10, AAV-11, AAV-12, AAV-13, AAV rh.74, and AAV-B1 and their derivatives. As mentioned above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. rAAVs with capsid mutations are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Modified capsids are also contemplated herein, including capsids with various post-translational modifications, such as glycosylation and deamidation. Deamidation of asparagine or glutamine side chains to convert asparagine residues to aspartic acid or isoaspartic acid residues, and conversion of glutamine to glutamic acid or isoglutamic acid are contemplated in the rAAV capsids provided herein. See, for example, Giles et al., Molecular Therapy, 26(12):2848-2862 (2018). Modified capsids are also contemplated herein to include targeting sequences that direct rAAV to diseased tissues and organs in need of treatment.

[0043] The DNA plasmids provided herein contain the rAAV genome described herein. The DNA plasmids can be introduced into cells permissive for infection by AAV helper viruses (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles using AAV9 capsid proteins. Techniques for producing rAAV, in which the rAAV genome to be packaged, rep and cap genes, and helper virus functions are provided in cells, are standard in the art. The production of rAAV particles requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, or may be derived from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, the entire disclosure of which is incorporated herein by reference. In various embodiments, AAV capsid proteins can be modified to enhance the delivery of recombinant rAAV. Modifications to capsid proteins are generally known in the art. For example, see US2005 / 0053922 and US2009 / 0202490, the entire disclosures of which are incorporated herein by reference.

[0044] A method for generating packaging cells is to create a cell line that stably expresses all the components necessary for rAAV production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene can be integrated into the genome of the cell. The rAAV genome can also be introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line can then be infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another non-limiting example of a suitable method uses adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cell.

[0045] The general principles of rAAV particle production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Ratschin et al., Mol. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis being placed on the portions of the documents relating to rAAV particle production.

[0046] Further provided herein are packaging cells that produce infectious rAAV particles. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 cells). In another embodiment, the packaging cells can be cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0047] Also provided herein are rAAVs (e.g., infectious, encapsidated rAAV particles) comprising the rAAV genome of the present disclosure. The genome of the rAAV lacks AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome. The rAAV genome can be a self-complementary (sc) genome. An rAAV having an sc genome is referred to herein as scAAV. The rAAV genome can be a single-stranded (ss) genome. An rAAV having a single-stranded genome is referred to herein as ssAAV.

[0048] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV from helper viruses are known in the art and can include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0049] Also provided are compositions comprising rAAV. The compositions comprise rAAV encoding a polypeptide of interest. The compositions may comprise two or more rAAV encoding different polypeptides of interest. In some embodiments, the rAAV is scAAV or ssAAV.

[0050] The compositions provided herein comprise rAAV and one or more pharmaceutically acceptable excipients. Acceptable excipients are non-toxic to recipients and preferably inert at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphates, e.g., phosphate-buffered saline (PBS), citrates, or other organic acids; antioxidants such as ascorbic acid; low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, copolymers such as Poloxamer 188, Pluronic® (e.g., Pluronic F68), or polyethylene glycol (PEG). The compositions provided herein can include a pharmaceutically acceptable aqueous excipient containing a non-ionic hypo-osmolar compound or contrast agent, such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the non-ionic hypo-osmolar compound can have one or more of the following properties: an osmolality of about 180 mg / mL, about 322 mOsm / kg water by vapor pressure osmometry, an osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20° C. and about 1.5 cp at 37° C., and a specific gravity of about 1.164 at 37° C. Exemplary compositions include about 20-40% non-ionic hypo-osmolar compound, or about 25% to about 35% non-ionic hypo-osmolar compound. An exemplary composition includes scAAV or rAAV viral particles formulated in 20 mM Tris (pH 8.0), 1 mM MgCl, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a non-ionic hypoosmolar compound. Another exemplary composition includes scAAV formulated in 1× PBS and 0.001% Pluronic F68.

[0051] Dosages may be expressed in units of viral genomes (vg). Dosages contemplated herein include those containing about 1 x 10 7 vg, approx. 1×10 8 vg, approx. 1×10 9 vg, approx. 5×10 9 vg, approx. 6×10 9 vg, approx. 7×10 9 vg, approx. 8×10 9 vg, approx. 9×10 9 vg, approx. 1×10 10 vg, approx. 2×10 10 vg, approx. 3×10 10 vg, approx. 4×10 10 vg, approx. 5×10 10 vg, approx. 1×10 11 vg, approx. 1.1×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.4×10 11 vg, approx. 1.5×10 11 vg, approx. 1.6×10 11 vg, approx. 1.7×10 11 vg, approx. 1.8×10 11 vg, approx. 1.9×10 11 vg, approx. 2×10 11 vg, approx. 3×10 11 vg, approx. 4×10 11 vg, approx. 5×10 11 vg, approx. 1×10 12 vg, approx. 1×10 13 vg, approx. 1.1×10 13 vg, approx. 1.2×10 13 vg, approx. 1.3×10 13 vg, approx. 1.5×10 13 vg, approx. 2×10 13 vg, approx. 2.5×10 13 vg, approx. 3×10 13 vg, approx. 3.5×10 13 vg, approx. 4×10 13 vg, approx. 4.5×10 13 vg, approx. 5×10 13vg, approx. 6×10 13 vg, approx. 1×10 14 vg, approx. 2×10 14 vg, approx. 3×10 14 vg, approx. 4×10 14 vg, approx. 5×10 14 vg, approx. 1×10 15 vg, approx. 1×10 16 vg, or more, of total viral genomes. Approximately 1 × 10 9 vg to approximately 1 × 10 10 vg, approx. 5×10 9 vg to about 5 × 10 10 vg, approx. 1×10 10 vg to approximately 1 × 10 11 vg, approx. 1×10 11 vg to approximately 1 × 10 15 vg, approx. 1×10 12 vg approx. 1×10 15 vg, approx. 1×10 12 vg to approximately 1 × 10 14 vg, approx. 1×10 13 vg to about 6 × 10 14 vg, and approximately 6 × 10 13 vg to approximately 1.0 × 10 14 vg, 2.0 × 10 14 vg, 3.0 × 10 14 vg, 5.0 × 10 14 Also contemplated are dosages of 1.65 x 10 11 vg.

[0052] Methods are provided for transducing target cochlear cells with rAAV, including inner hair cells, outer hair cells, ganglion cells, supporting cells, Deiters cells, columnar cells, and epithelial cells.

[0053] The term "transduction" refers to the administration / delivery of a CLN6 polynucleotide to target cells, either in vivo or in vitro, via a replication-deficient rAAV of the present disclosure, resulting in expression of a functional polypeptide by the recipient cells. Transduction of cells with the rAAV of the present disclosure results in sustained expression of the polypeptide or RNA encoded by the rAAV. Thus, the present disclosure provides a method for administering / delivering an rAAV encoding a polypeptide encoded by a transgene to a subject by intrathecal or IV delivery, or any combination thereof. Intrathecal delivery refers to delivery to the subarachnoid space of the brain or spinal cord. In some embodiments, intrathecal administration is by intracisternal administration.

[0054] Disorders related to hearing loss The present disclosure provides methods for treating hearing loss. Conductive hearing loss results from abnormalities in the ossicles of the outer and / or middle ear. Sensorineural hearing loss results from dysfunction of inner ear structures (i.e., the cochlear nerve or auditory nerve). Mixed hearing loss is a combination of conductive and sensorineural hearing loss. Central auditory dysfunction results from damage or dysfunction at the level of the eighth cranial nerve, auditory brainstem, or cerebral cortex.

[0055] Hereditary hearing loss is subdivided into Mendelian, which includes both syndromic and nonsyndromic cases, or complex inheritance, which includes both genetic and environmental factors. A summary of the genetic causes of syndromic hearing loss is shown in Table 1. A summary of the genetic causes of nonsyndromic hearing loss is shown in Tables 2 and 3. A summary of X-linked nonsyndromic hearing loss is shown in Table 4. (See Shearer AE, Hildebrand MS, Smith RJH. Hereditary Hearing Loss and Deafness Overview. 1999 Feb 14 [Updated 2017 Jul 27]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2019.) [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4]

[0056] Mitochondrial DNA pathogenic variants are implicated in a variety of disorders associated with hearing loss, ranging from rare neuromuscular syndromes such as Kearns-Sayre syndrome, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myocardial epilepsy with irregular red fibers (MERRF), and neurogenic weakness with ataxia and retinitis pigmentosa (NARP) to common conditions such as diabetes, Parkinson's disease, and Alzheimer's disease. For example, a typical pathogenic variant associated with hearing loss in patients with diabetes is the 3243A-to-G transition in MTTL1 and MELAS. A summary of mitochondrial DNA mutations associated with hearing loss is shown in Table 5. (See Shearer AE, Hildebrand MS, Smith RJH. Hereditary Hearing Loss and Deafness Overview. 1999 Feb 14 [Updated 2017 Jul 27]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2019.) [Table 5]

[0057] In any of the disclosed methods, treating hearing loss in a subject in need results in an improvement in the subject's hearing or an improvement or reduction in the subject's hearing impairment. Tests to determine whether the treatment methods described herein improve or reduce hearing loss or hearing impairment include physiological tests that objectively determine the functional status of the auditory system and audiometry, which subjectively determine how an individual processes auditory information. Physiological tests include auditory brainstem response testing (ABR), which uses stimuli (e.g., clicks) to evoke electrophysiological responses originating from the eighth cranial nerve and auditory brainstem and recorded with surface electrodes. The ABR's "wave V detection threshold" best correlates with hyperacusis in the 1500-4000 Hz region in neurologically normal individuals; the ABR does not assess sensitivity at low frequencies (<1500 Hz).

[0058] Auditory steady-state response (ASSR) testing uses an objective, statistically-based mathematical detection algorithm to detect and define hearing thresholds. ASSRs can be obtained using broadband or frequency-specific stimuli and can provide derivatives of hearing thresholds across the severe-to-severe range. ASSR testing is frequently used to provide frequency-specific information that ABRs do not. Test frequencies of 500, 1000, 2000, and 4000 Hz are commonly used.

[0059] Evoked otoacoustic emissions (EOAEs) are sounds generated within the cochlea and are measured in the ear canal using a probe equipped with a microphone and transducer. EOAEs primarily reflect the activity of the outer hair cells in the cochlea over a wide frequency range and are present in ears with hearing sensitivity better than 40–50 dB HL. Immittance tests (tympanometry, acoustic reflex threshold, acoustic reflex attenuation) assess the peripheral auditory system, including middle ear pressure, tympanic membrane mobility, Eustachian tube function, and middle ear bone mobility.

[0060] Hearing tests include behavioral tests such as behavioral observational audiometry (BOA) and visual reinforcement audiometry (VRA). Pure-tone audiometry (air and bone conduction) involves determining the minimum intensity at which an individual "hears" a pure tone as a function of frequency (or pitch). Octave frequencies from 250 (near middle C) to 8000 Hz are tested using earphones. Intensity, or loudness, is measured in decibels (dB) and is defined as the ratio of two sound pressures. 0 dB HL is the average threshold for adults with normal hearing, and 120 dB HL is a sound loud enough to cause pain. Speech reception thresholds (SRT) and speech discrimination are assessed. In air conduction audiometry, sounds are presented through earphones, and thresholds vary depending on the condition of the ear canal, middle ear, and inner ear. In bone conduction audiometry, audiometry sounds are presented through a vibrator placed on the mastoid bone or forehead, thereby bypassing the outer and middle ear, and thresholds vary depending on the condition of the inner ear. Additional testing includes conditional hearing testing, which creates a full frequency-specific audiogram for each ear, and traditional hearing testing, which shows when an individual hears sounds.

[0061] An audioprofile refers to the recording of multiple audiograms on a single graph. These audiograms can be from a single individual at different times, but often from different members of the same family, who usually segregate hearing loss in an autosomal dominant manner. By plotting multiple audiograms with age on the same graph, it is possible to understand the progression of age-related hearing loss within these families.

[0062] Transgene The disclosed method includes delivering any transgene of interest to cochlear cells. The transgene is a polynucleotide sequence encoding a polypeptide of interest, or a nucleic acid that inhibits, suppresses, or silences the expression of the gene of interest, such as siRNA or miRNA. Exemplary transgenes are human atonal transcription factor (ATOH1) cDNA (SEQ ID NO: 2), otoferlin cDNA (SEQ ID NO: 4), gap junction protein beta 2 (GJB2) cDNA (SEQ ID NO: 6), SLC264 cDNA (SEQ ID NO: 8), forkhead box O3 (FOXO3) cDNA (SEQ ID NO: 10), activin A cDNA (SEQ ID NO: 12), and follistatin cDNA (SEQ ID NO: 14).

[0063] Exemplary transgenes are polynucleotides encoding human atonal transcription factor (SEQ ID NO: 3), otoferlin (SEQ ID NO: 5), gap junction protein beta 2 (SEQ ID NO: 7), pendrin (SEQ ID NO: 9), forkhead box 1 (SEQ ID NO: 11), activin A or inhibin (SEQ ID NO: 13), follistatin (SEQ ID NO: 15), galectin-1 (SEQ ID NO: 20), and galectin-3 (SEQ ID NO: 22) proteins.

[0064] Cochlear-expressed miRNAs shRNAs and miRNAs are contemplated as transgenes included in the disclosed optimized gene therapy vectors.

[0065] The rAAV genomes provided herein can include the wild-type nucleic acid sequence of any of the genes provided herein in Table 1, Table 2, Table 3, Table 4, or Table 5. Additionally, the rAAV genomes provided herein can include the cDNA sequence of one of the following: human atonal transcription factor (ATOH1) cDNA (SEQ ID NO: 2), otoferlin cDNA (SEQ ID NO: 4), gap junction protein beta 2 (GJB2) cDNA (SEQ ID NO: 6), SLC264 cDNA (SEQ ID NO: 8), forkhead box 1 (FOXO3) cDNA (SEQ ID NO: 10), activin A cDNA (SEQ ID NO: 12), follistatin (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21) cDNA. For example, a polypeptide encoded by a transgene includes a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the transgene sequence.

[0066] For example, the rAAV genomes provided herein include polynucleotides encoding human atonal transcription factor (SEQ ID NO:3), otoferlin (SEQ ID NO:5), gap junction protein beta 2 (SEQ ID NO:7), pendrin (SEQ ID NO:9), forkhead box 1 (SEQ ID NO:11), activin A or inhibin (SEQ ID NO:13), follistatin (SEQ ID NO:15), galectin-1 (SEQ ID NO:20), or galectin-3 (SEQ ID NO:22). Polypeptides include polypeptides comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the transgene sequence and that retains a desired activity.

[0067] In some cases, the rAAV genomes provided herein include a polynucleotide that encodes a polypeptide or is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence that encodes a polypeptide having a desired activity.

[0068] The rAAV genomes provided herein, in some embodiments, comprise a transgene comprising a polynucleotide sequence that encodes a polypeptide having a desired activity and that hybridizes under stringent conditions to any one of the nucleic acid sequences of known transgenes of interest, or their complements.

[0069] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing include, but are not limited to, 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989).

[0070] Administration method Intrathecal administration is exemplified herein. These methods include transducing target cells with one or more rAAVs described herein. In some embodiments, rAAV viral particles containing a transgene are administered or delivered to the ear, brain, and / or spinal cord of a patient. In some embodiments, polynucleotides are delivered to the spinal cord. Brain regions contemplated for delivery include, but are not limited to, the motor cortex, visual cortex, cerebellum, and brainstem. In some embodiments, polynucleotides are delivered to the spinal cord. In some embodiments, polynucleotides are delivered to lower motor neurons. Polynucleotides can be delivered to cochlear cells, such as inner hair cells, outer hair cells, ganglion cells, supporting cells, Deiters cells, columnar cells, and epithelial cells.

[0071] In some embodiments of the methods provided herein, the patient is maintained in the Trendelenberg position (head down) after administration of the rAAV (e.g., for about 5, about 10, about 15, or about 20 minutes). For example, the patient may be tilted in a head-down position (about 1 to about 30 degrees, about 15 to about 30 degrees, about 30 to about 60 degrees, about 60 to about 90 degrees, or about 90 to about 180 degrees).

[0072] For intraventricular injection, a needle is inserted into the skull and the fluid is injected into the cavity containing the cerebrospinal fluid. For example, intraventricular injections are performed by clinically trained surgeons using methods known in the art.

[0073] The methods provided herein include administering an effective dose or effective doses of a composition comprising the rAAV provided herein to a subject (e.g., an animal, including, but not limited to, a human patient) in need thereof. If the dose is administered before the onset of symptoms of a hearing loss-related disorder, the administration is prophylactic. If the dose is administered after the onset of symptoms of a hearing loss-related disorder, the administration is therapeutic. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with a hearing loss-related disorder, delays or prevents the progression of the disorder, reduces the extent of the disorder, results in remission (partial or complete) of the disorder, and / or prolongs hearing and / or survival. Compared to pre-treatment subjects or compared to untreated subjects, the methods provided herein result in stabilization, a reduction in the progression of hearing loss, or improvement in tone. [Example]

[0074] The following examples illustrate particular embodiments, but it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0075] Example 1 Production of scAAV9.GFP Human GFP cDNA clones were obtained from Origene, Rockville, MD. The GFP cDNA alone or the GFP cDNA and shSOD-1 cDNA were further subcloned into the self-complementary AAV9 genome under the hybrid chicken β-actin promoter (CB). The plasmid constructs also contained one or more of the following: the CB promoter (SEQ ID NO: 1), an intron such as the simian virus 40 (SV40) chimeric intron, and the bovine growth hormone (BGH) polyadenylation signal (BGH polyA). A schematic diagram of the plasmid construct, designated AAV9.CB.GFP, showing the GFP cDNA inserted between the AAV2 ITRs is provided in Figure 1. The AAV9.CB.shSOD1.GFP plasmid contains the human SOD-1 cDNA inserted between the SV40 intron and the GFP cDNA. The constructs were packaged into either AAV9 genome.

[0076] Example 2 Intrathecal AAV9 delivery and targeting of cochlear cells The mouse is 1.65 x 10 11 vg or 3.3×10 11 Mice received intrathecal (IT) injections of either scAAV9.shSOD1.CB.GFP or scAAV9.CB.GFP (vg). scAAV9.shSOD1.CB.GFP or scAAV9.CB.GFP was formulated in 1x PBS and 0.001% Pluronic F68 (denoted as PBS / F68). Mice were euthanized 3 weeks after injection. Cochleae from treated mice were cryosectioned and stained for GFP. Sections were also stained with DAPI to demonstrate cell location.

[0077] Figures 2-5 show data from mice injected with scAAV9.shSOD1.CB.GFP. Figure 2 shows GFP staining in sagittal cryosections of IT-treated cochleae. The hematoxylin and eosin staining is provided as a reference and is not a photograph of the cochlea from the injected mouse (shx2IT-LFLR). The data demonstrate strong GFP staining throughout the mouse cochlea after IT injection. Figure 3 shows staining from four different mice (shx2IT-LF, shx2IT-2F, shx2IT-LE, shx2IT-LFLR). This figure demonstrates widespread GFP expression and a uniform pattern of GFP expression in all injected mice. Cells of the organ of Corti express high levels of GFP. Figure 4 shows various regions of the mouse cochlea in which GFP was expressed after IT injection. In Figure 5, sagittal cryosections of the cochlea from an IT-injected mouse (shx2IT-LE) were stained with actin in addition to GFP and DAPI. This figure shows that targeting of inner hair cells (IHCs) was successful, as they express the GFP transgene in several organs of Corti. The figure shows a zoom-in to increase magnification to clearly see the IHC cells.

[0078] Figures 6-8 show data from mice injected with scAAV9.CB.GFP. Cochleae from IT-injected mice were also sectioned coronally with a vibratome for different views of GFP expression patterns. Figures 6 and 7 show that inner hair cells within the cochlea strongly stain for GFP in the injected mice (mouse #2 and mouse #0). These figures also demonstrate that IT injection results in broad localization of GFP expression. Figure 8 shows the vestibule of an IT-injected mouse stained with DAPI, GFP, and actin. These figures demonstrate the targeting of inner hair cells throughout the cochlea across the various turns where various wavelengths are treated.

[0079] Example 3 Transduction of mouse cochlea after intrathecal delivery and exposure to noise Stress on hair cells in the mouse inner ear was induced by exposing the mice to noise at a sound pressure level of 100 dB for 2 hours. After noise exposure, mice were administered scAAV9.CB.GFP by a single intrathecal injection (IT) either immediately after exposure (0 h.pi) or 24 hours after exposure (24 h.pi). Mice were administered 1.65 × 10 11 Each mouse was injected with 1000 mg of scAAV9.CB.GFP. scAAV9.CB.GFP was formulated in 1x PBS and 0.001% Pluronic F68 (denoted as PBS / F68). Three weeks after injection, the mouse was euthanized.

[0080] To examine cochlear expression of the transgene, immunohistochemical analysis was performed to visualize the GFP protein. Cross sections of the organ tissue were stained for Dapi, GFP, and phalloidin, which stain actin filaments, or for myosin-VIIa or myo7A. Staining for myo7A was performed specifically to label hair cells. As shown in Figures 9-12, inner hair cells expressed the transgene GFP after IT injection of scAAV9.CB.GFP immediately after exposure to noise.

[0081] Figures 13-16 show GFP and myo7A staining, demonstrating that GFP expression was detected when IT injection was performed 24 h after noise exposure, although expression levels were generally low in inner hair cells. Compared to GFP expression when IT injection was performed immediately after noise exposure, GFP expression was lower when IT injection was delayed 24 h after noise exposure. Figures 16 and 17 demonstrate that administering scAAV9.CB.GFP 24 h after injury weakens the GFP signal, but targeting of inner hair cells still occurs even with delayed administration. Figure 16 shows an image of an IT-injected mouse imaged at optimal exposure at 24 hpi. This photograph demonstrates that GFP expression in the intrathecally injected animal at 24 hpi is lower than that in the intrathecally injected animal at 0 hpi, due to the imaging at optimal exposure at 24 hpi overexposing the animal at 0 hpi. The hematoxylin and eosin staining shown in Figure 17 demonstrates that inner hair cells are not damaged by the noise injury protocol used in this study.

[0082] Example 4 Transduction of mouse cochlea after intravenous injection scAAV9.CB.GFP was also administered to normal mice via a single tail vein injection. Mice received 1.65 × 10 12 Mice were injected with 1000 mg / kg of scAAV9.CB.GFP. scAAV9.CB.GFP was formulated in 1x PBS and 0.001% Pluronic F68 (denoted as PBS / F68). Three weeks after injection, mice were euthanized. As shown in Figure 18, preliminary data provided here indicate that scAAV9.CB.GFP delivered GFP to the cochlea but did not transduce inner hair cells or dorsal root ganglion neurons when administered using tail vein injection. Instead, transduction appears to remain around the blood vessels.

[0083] Subsequent studies have shown that 1.65 × 10 12scAAV9.CB.GFP was administered to normal mice via a single tail vein injection, and the mice were euthanized 3 weeks after injection. The immunohistochemistry data shown in Figures 19-22 demonstrate that intravenous injection did not result in transduction of inner hair cells in healthy cochleae. Figure 23 shows data from uninjected control mice exposed to noise injury. In certain embodiments, for example, the following items are provided: (Item 1) A method for delivering a transgene to cochlear cells of a subject, comprising administering a gene therapy vector encoding the transgene, wherein the gene therapy vector is administered to the subject using intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 2) Item 10. The method of item 1, wherein the cochlear cells are inner hair cells. (Item 3) A method for treating hearing loss or a hearing loss-related disorder in a subject, comprising administering to the subject a gene therapy vector encoding a transgene, wherein the gene therapy vector is administered using intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 4) 4. The method of item 3, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejjerg syndrome. (Item 5) 3. The method of item 1 or 2, wherein the subject suffers from hearing loss or a hearing loss-related disorder. (Item 6) 6. The method of item 5, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejjerg syndrome. (Item 7) 7. The method of any one of items 1 to 6, wherein the transgene encodes human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21). (Item 8) 7. The method of any one of items 1 to 6, wherein the transgene is an miRNA or siRNA against human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21). (Item 9) 9. The method of any one of items 1 to 8, wherein the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT or Anc80, AAV7m8, or a derivative thereof. (Item 10) 10. The method of any one of items 1 to 9, wherein the gene therapy vector is administered using intrathecal delivery, and the method further comprises placing the subject in Trendelenburg position after administration of the gene therapy vector. (Item 11) 10. The method of any one of items 1 to 9, wherein the gene therapy vector is administered using a delivery method that applies the vector directly to the cerebrospinal fluid. (Item 12) A composition for delivering a transgene to cochlear cells of a subject, the composition comprising a gene therapy vector encoding the transgene, the composition formulated for intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 13) Item 2. The composition of item 1, wherein the cochlear cells are inner hair cells. (Item 14) A composition for treating hearing loss or a hearing loss-related disorder in a subject, said composition comprising a gene therapy vector encoding a transgene, said composition formulated for intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 15) 15. The composition of item 14, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejäger syndrome. (Item 16) 14. The composition of item 12 or 13, wherein the subject is suffering from hearing loss or a hearing loss-related disorder. (Item 17) 17. The composition of item 16, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neurocytopathy syndrome, or Mohr-Tranejjerg syndrome. (Item 18) 18. The composition of any one of Items 12 to 17, wherein the transgene encodes human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21). (Item 19) 18. The composition of any one of Items 12 to 17, wherein the transgene is an miRNA or siRNA against human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21). (Item 20) 20. The composition of any one of items 12 to 19, wherein the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT or Anc80, AAV7m8, or a derivative thereof. (Item 21) 21. The composition of any one of items 12 to 20, wherein the composition is formulated for intrathecal delivery to a subject, and the subject is placed in the Trendelenburg position after administration of the composition. (Item 22) 21. The composition of any one of items 12 to 20, formulated for administration using a delivery method that applies the vector directly to the cerebrospinal fluid. (Item 23) Use of a gene therapy vector for preparing a drug for delivering a transgene to cochlear cells of a subject, wherein the gene therapy vector encodes the transgene, and wherein the drug is formulated for administration to the subject using intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 24) 24. The use according to item 23, wherein the cochlear cells are inner hair cells. (Item 25) Use of a gene therapy vector for the preparation of a medicament for treating hearing loss or a hearing loss-related disorder in a subject, wherein the gene therapy vector encodes a transgene and the medicament is formulated for intravenous delivery, intrathecal delivery, or any delivery method that accesses the cerebrospinal fluid. (Item 26) 26. The use of item 25, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejjerg syndrome. (Item 27) 25. The use of item 23 or 24, wherein the subject suffers from hearing loss or a hearing loss-related disorder. (Item 28) 28. The use of item 27, wherein the hearing loss related disorder is Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, deafness-dystonia-optic neuropathic syndrome, or Mohr-Tranejjerg syndrome. (Item 29) 29. The use according to any one of Items 23 to 28, wherein the transgene encodes human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21). (Item 30) 29. The use according to any one of Items 23 to 28, wherein the transgene is an miRNA or siRNA against human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), or follistatin (FST) (SEQ ID NO: 14). (Item 31) 31. The use of any one of items 23 to 30, wherein the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT or Anc80, AAV7m8, or a derivative thereof. (Item 32) 32. The use of any one of items 23 to 31, wherein the agent is formulated for intrathecal delivery and the subject is placed in Trendelenburg position after administration of the agent. (Item 33) 32. The use according to any one of items 23 to 31, wherein the agent is formulated for a delivery method in which the vector is applied directly to the cerebrospinal fluid.

Claims

1. 1. A composition for delivering a transgene to cochlear cells of a subject, the composition comprising a gene therapy vector encoding the transgene, the gene therapy vector being AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, Anc80, or AAV7m8, the composition being administered by intrathecal delivery or any delivery method that directly accesses cerebrospinal fluid, and the cochlear cells being inner hair cells.

2. 1. A composition for treating hearing loss in a subject, the composition comprising a gene therapy vector encoding a transgene, the composition being administered by intrathecal delivery or any delivery method that directly accesses cerebrospinal fluid, the gene therapy vector being AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, Anc80, or AAV7m8, and the transgene being delivered to inner hair cells.

3. 3. The composition of claim 2, wherein the subject is suffering from Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, or Mohr-Tranejäger syndrome.

4. The composition of claim 1 , wherein the subject is suffering from hearing loss.

5. 5. The composition of claim 4, wherein the subject is suffering from Waardenburg syndrome (WS), branchio-oto-renal spectrum disorder, neurofibromatosis 2 (NF2), Stickler syndrome, Usher syndrome type I, Usher syndrome type II, Usher syndrome type III, Pendred syndrome, Jervell-Lange-Nielsen syndrome, biotinidase deficiency, Refsum disease, Alport syndrome, or Mohr-Tranejäger syndrome.

6. The transgene may be a human atonal transcription factor (ATOH1) (SEQ ID NO: 2), otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin ( 2. The composition of claim 1, wherein the composition encodes a nucleotide sequence encoding ...

7. 2. The composition of claim 1, wherein the transgene is a miRNA or siRNA against otoferlin (SEQ ID NO: 4), gap junction protein beta 2 (SEQ ID NO: 6), pendrin (SLC26A) (SEQ ID NO: 8), forkhead box 1 (FOXG1) (SEQ ID NO: 10), activin A or inhibin (SEQ ID NO: 12), follistatin (FST) (SEQ ID NO: 14), galectin-1 (SEQ ID NO: 19), or galectin-3 (SEQ ID NO: 21).

8. 8. The composition of any one of claims 1 to 7, wherein the composition is administered to the subject by intrathecal delivery, and the subject is placed in the Trendelenburg position after administration of the composition.

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