Myoglobulin 15 promoters and uses thereof

Abstract

The present disclosure relates to myosin 15 promoters and uses thereof. The present disclosure provides polynucleotides containing a region of a myosin 15 (Myo15) promoter, which can be used to promote specific expression of a transgene in hair cells, as well as vectors containing the polynucleotides. The polynucleotides described herein can be operably linked to a transgene, such as a transgene encoding a therapeutic protein, in order to promote hair cell-specific expression of the transgene. The polynucleotides described herein can be operably linked to a therapeutic transgene and used to treat a subject having or at risk of developing hearing loss or vestibular dysfunction.

Description

[0001] This application is a divisional application. The filing date of the original application is February 7, 2020, the application number is 202080026444.6 (PCT / US2020 / 017292), and the name of the invention is “Myosin 15 promoter and its use”.

[0002] Sequence Listing

[0003] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on February 7, 2020 is named 51471-002WO4_Sequence_Listing_2.7.20_ST25 and is 26,901 bytes in size. Technical Field

[0004] Described herein are polynucleotides containing a region of the myosin 15 (Myo15) promoter, and vectors comprising the polynucleotides, which can be used to promote expression of a transgene in hair cells (e.g., cochlear hair cells, such as inner hair cells and outer hair cells, and / or vestibular hair cells). Also disclosed are methods of using the polynucleotides and vectors of the invention to achieve expression of a transgene in hair cells to treat hearing loss and / or vestibular dysfunction. Background Art

[0005] Hearing loss is a major public health problem, estimated to affect nearly 15% of school-age children and one-third of people aged 65 years. The most common type of hearing loss is sensorineural hearing loss, a type of hearing loss caused by defects in inner ear cells (such as cochlear hair cells) or in the neural pathways that extend from the inner ear to the brain. Sensorineural hearing loss is usually acquired and has a variety of causes, including acoustic trauma, disease or infection, head trauma, ototoxic drugs, and aging. Sensorineural hearing loss also has genetic causes, such as mutations in genes involved in the development and function of the inner ear. Mutations in more than 90 such genes have been identified, including mutations inherited in autosomal recessive, autosomal dominant, and X-linked patterns.

[0006] Factors that disrupt the development, survival, or integrity of cochlear hair cells, such as genetic mutations, disease or infection, ototoxic drugs, head trauma, and aging, can similarly affect vestibular hair cells and are therefore also associated with vestibular dysfunction, including vertigo, dizziness, and imbalance. Indeed, patients carrying mutations that disrupt hair cell development or function can experience both hearing loss and vestibular dysfunction, or either condition alone. In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a possible solution; however, there are still few methods that specifically target hair cells, which are often associated with hearing loss and vestibular dysfunction. New therapies that target hair cells to treat sensorineural hearing loss or vestibular dysfunction are needed. Summary of the invention

[0007] The present invention provides compositions and methods for promoting expression of target genes, such as genes that promote or improve hair cell function or survival, in specific cell types. The compositions and methods described herein relate to polynucleotides that stimulate transcription of transgenes in hair cells of the inner ear (e.g., cochlear hair cells and vestibular hair cells). The polynucleotides described herein can be operably linked to a therapeutic transgene and can be administered to a patient to treat or prevent hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance).

[0008] In a first aspect, the present invention provides a polynucleotide comprising a first region linked (e.g., operably linked) to a second region, the first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 1 or a functional part or derivative thereof comprising SEQ ID NO: 3 and / or SEQ ID NO: 4, the second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 2 or a functional part or derivative thereof comprising SEQ ID NO: 8 and / or SEQ ID NO: 9, the polynucleotide optionally containing 1 to 100 nucleotides (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35) located between the first region and the second region. , 1-40, 1-45, 1-50, 1-60, 1-70, 1-80, 1-90, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90 or 20-100 nucleotides).

[0009] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO: 1.

[0010] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:2.

[0011] In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 16. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 36. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 37.

[0012] In another aspect, the present invention provides a polynucleotide comprising a first region linked (e.g., operably linked) to a second region, the first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof comprising SEQ ID NO: 8 and / or SEQ ID NO: 9, the second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof comprising SEQ ID NO: 3 and / or SEQ ID NO: 4, the polynucleotide optionally containing 1 to 100 nucleotides (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35) located between the first region and the second region. , 1-40, 1-45, 1-50, 1-60, 1-70, 1-80, 1-90, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90 or 20-100 nucleotides).

[0013] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:2.

[0014] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:1.

[0015] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 35.

[0016] On the other hand, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO:1 or a functional portion or derivative thereof comprising SEQ ID NO:3 and / or SEQ ID NO:4.

[0017] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:1.

[0018] On the other hand, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO:2 or a functional portion or derivative thereof comprising SEQ ID NO:8 and / or SEQ ID NO:9.

[0019] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:2.

[0020] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:3. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:4. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:3 and the sequence of SEQ ID NO:4. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:5. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:6. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:7. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:27.

[0021] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:8. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:9. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:9. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:28. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:29. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:10. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:11. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:12. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:32.

[0022] In another aspect, the present invention provides a polynucleotide comprising a first region linked (e.g., operably linked) to a second region, the first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 17 or a functional portion or derivative thereof comprising SEQ ID NO: 19, the second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 18 or a functional portion or derivative thereof comprising SEQ ID NO: 20 and / or SEQ ID NO: 21, the polynucleotide optionally containing 1 to 400 nucleotides (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35) located between the first region and the second region. , 1-40, 1-45, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-125, 1-150, 1-175, 1-200, 1-225, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-100, 40-100, 50-100, 50-150, 50-200, 50-250, 50-300, 50-350, 50-400, 100-150, 100-200, 10 or 350-400 nucleotides).

[0023] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:17.

[0024] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:18.

[0025] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:25.

[0026] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:26.

[0027] In another aspect, the present invention provides a polynucleotide comprising a first region linked (e.g., operably linked) to a second region, the first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 18 or a functional part or derivative thereof comprising SEQ ID NO: 20 and / or SEQ ID NO: 21, the second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 17 or a functional part or derivative thereof comprising SEQ ID NO: 19, the polynucleotide optionally containing 1 to 400 nucleotides (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35) located between the first region and the second region. , 1-40, 1-45, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-125, 1-150, 1-175, 1-200, 1-225, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-100, 40-100, 50-100, 50-150, 50-200, 50-250, 50-300, 50-350, 50-400, 100-150, 100-200, 10 or 350-400 nucleotides).

[0028] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:18.

[0029] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:17.

[0030] In another aspect, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO:17 or a functional portion or derivative thereof comprising a sequence of SEQ ID NO:19.

[0031] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:17.

[0032] On the other hand, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO:18 or a functional portion or derivative thereof comprising a sequence of SEQ ID NO:20 and / or SEQ ID NO:21.

[0033] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:18.

[0034] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:17 contains the sequence of SEQ ID NO:19.

[0035] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 20. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 21. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 22. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 23. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO: 18 contains the sequence of SEQ ID NO: 24.

[0036] In another aspect, the present invention provides a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the nucleic acid sequence of any one of SEQ ID NOs: 27-35. In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 28. In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 32. In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO: 34.

[0037] In some embodiments of any of the aforementioned aspects, the polynucleotide, when operably linked to a transgene and introduced into hair cells, induces expression of the transgene.

[0038] In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide of the present invention. In some embodiments, the polynucleotide is operably linked to a transgene. In some embodiments, the transgene comprises a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the polynucleotide is capable of directing hair cell-specific expression of a therapeutic protein by the nucleic acid sequence in mammalian hair cells. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the cochlear hair cells are inner hair cells. In some embodiments, the cochlear hair cells are outer hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0039] In some embodiments, the therapeutic protein is selected from the group consisting of 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, C ABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, K CNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1 and BIP.

[0040] In some embodiments, the nucleic acid vector is a plasmid, a cosmid, an artificial chromosome or a viral vector. In some embodiments, the nucleic acid vector is a viral vector selected from the group consisting of: adeno-associated virus (AAV), adenovirus and lentivirus. In some embodiments, the viral vector is an AAV vector. In some embodiments, the serotype of the AAV vector is selected from the group containing: 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. In some embodiments, the serotype of the AAV vector is AAV1. In some embodiments, the serotype of the AAV vector is AAV9. In some embodiments, the serotype of the AAV vector is AAV6. In some embodiments, the serotype of the AAV vector is Anc80. In some embodiments, the serotype of the AAV vector is Anc80L65. In some embodiments, the serotype of the AAV vector is DJ / 9. In some embodiments, the serotype of the AAV vector is 7m8. In some embodiments, the serotype of the AAV vector is AAV2. In some embodiments, the serotype of the AAV vector is AAV2quad (YF). In some embodiments, the serotype of the AAV vector is PHP.B. In some embodiments, the serotype of the AAV vector is AAV8.

[0041] In another aspect, the present invention provides a composition comprising the nucleic acid vector of the present invention. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0042] In another aspect, the present invention provides a method for increasing the expression of a therapeutic protein in mammalian hair cells, the method being achieved by contacting the mammalian hair cells with a nucleic acid vector of the present invention or a composition of the present invention. In some embodiments, the expression of the therapeutic protein is specifically increased in hair cells.

[0043] In some embodiments, the mammalian hair cells are human hair cells.

[0044] In some embodiments, the mammalian hair cell is a cochlear hair cell. In some embodiments, the cochlear hair cell is an inner hair cell. In some embodiments, the cochlear hair cell is an outer hair cell.

[0045] In some embodiments, the mammalian hair cells are vestibular hair cells.

[0046] In some embodiments, expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells.

[0047] In another aspect, the present invention provides a method of treating a subject having hearing loss (e.g., sensorineural hearing loss) or at risk of developing hearing loss by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0048] In some embodiments, the hearing loss is hereditary hearing loss. In some embodiments, the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

[0049] In some embodiments, the hearing loss is acquired hearing loss. In some embodiments, 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 hearing loss caused by ototoxic drugs. In some embodiments, the acquired hearing loss is age-related hearing loss. In some embodiments, the hearing loss is noise-induced hearing loss. In some embodiments, the hearing loss is hearing loss caused by ototoxic drugs.

[0050] On the other hand, the present invention provides a method for treating a subject suffering from vestibular dysfunction or having a risk of developing vestibular dysfunction, the method being achieved by administering an effective amount of a nucleic acid vector of the present invention or a composition of the present invention to the subject. In some embodiments, the vestibular dysfunction is vertigo, dizziness or imbalance (e.g., balance disorder).

[0051] In another aspect, the present invention provides a method for promoting hair cell regeneration in a subject in need thereof, the method being achieved by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0052] On the other hand, the present invention provides a method for preventing or reducing hair cell damage or death caused by ototoxic drugs, the method being achieved by administering an effective amount of a nucleic acid vector of the present invention or a composition of the present invention to the subject. In some embodiments, the ototoxic drug is selected from the group comprising: aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin and amikacin), antineoplastic agents (e.g., platinum-containing chemotherapeutics such as cisplatin, carboplatin and oxaliplatin), ethacrynic acid, furosemide, salicylates (e.g., aspirin, particularly at high doses) and quinine. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0053] In another aspect, the present invention provides a method for treating a subject suffering from tinnitus, the method being achieved by administering an effective amount of the nucleic acid vector of the present invention or the composition of the present invention to the subject.

[0054] In some embodiments of any of the preceding aspects, the hearing loss, vestibular dysfunction, or tinnitus is associated with a loss of hair cells (eg, cochlear and / or vestibular hair cells).

[0055] In another aspect, the present invention provides a method for preventing or reducing hair cell damage or death in a subject in need thereof, the method being achieved by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0056] In another aspect, the present invention provides a method of increasing hair cell survival in a subject in need thereof, the method being achieved by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0057] In some embodiments of any of the preceding aspects, the hair cells are cochlear hair cells. In some embodiments of any of the preceding aspects, the cochlear hair cells are inner hair cells. In some embodiments of any of the preceding aspects, the cochlear hair cells are outer hair cells. In some embodiments of any of the preceding aspects, the mammalian hair cells are vestibular hair cells.

[0058] In some embodiments of any of the preceding aspects, the method further comprises a step of assessing the subject's hearing prior to administering the nucleic acid vector or composition (e.g., assessing hearing using a standard test such as audiometry, auditory brainstem response (ABR), electrochocleography (ECOG), or otoacoustic emissions).

[0059] In some embodiments of any of the preceding aspects, the method further comprises a step of assessing the subject's hearing after administering the nucleic acid vector or composition (e.g., assessing hearing using a standard test such as audiometry, ABR, ECOG, or otoacoustic emissions).

[0060] In some embodiments of any of the foregoing aspects, the method further comprises a step of assessing the subject's vestibular function prior to administering the nucleic acid vector or composition (e.g., assessing vestibular function using a standard test such as an electronystagmogram (ENG) or videonystagmogram (VNG), posturography, rotary-chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), or a specialized clinical balance test).

[0061] In some embodiments of any of the foregoing aspects, the method further comprises a step of assessing the subject's vestibular function prior to administering the nucleic acid vector or composition (e.g., assessing vestibular function using a standard test such as ENG or VNG, posturography, swivel chair test, ECOG, VEMP, or a specialist clinical balance test).

[0062] In some embodiments of any of the preceding aspects, the nucleic acid vector or composition is administered topically (eg, to the inner ear, eg, into the perilymph or endolymph, such as through the oval window, round window, or horizontal semicircular canal).

[0063] In some embodiments of any of the preceding aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce vestibular dysfunction, prevent or reduce tinnitus, delay the development of hearing loss, delay the development of vestibular dysfunction, slow the progression of hearing loss, slow the progression of vestibular dysfunction, improve hearing, improve vestibular function, improve hair cell function, prevent or reduce hair cell damage, prevent or reduce hair cell death, or increase the number of hair cells.

[0064] In some embodiments of any of the preceding aspects, the subject is a human.

[0065] In another aspect, the present invention provides a kit comprising the nucleic acid vector of the present invention or the composition of the present invention.

[0066] definition

[0067] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0068] As used herein, "administering" refers to providing or administering a therapeutic agent (eg, a nucleic acid vector containing a myosin 15 (Myo15) promoter operably linked to a transgene) to a subject by any effective route. Exemplary routes of administration are described below.

[0069] As used herein, the term "cell type" refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of common cell types may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of common cell types may include those separated from common tissues (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those separated from other structures and / or regions of common organs, tissue systems, blood vessels, or organisms.

[0070] 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 associated with hearing loss and deafness.

[0071] As used herein, the terms "conservative mutation", "conservative substitution" and "conservative amino acid substitution" refer to substitution of one or more amino acids into one or more different amino acids that exhibit similar physicochemical properties (such as polarity, electrostatic charge and steric bulk). These properties for each of the 20 naturally occurring amino acids are summarized in Table 1 below.

[0072] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0073]

[0074]

[0075] It will be appreciated from this table that the conservative amino acid family includes (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is one in which an amino acid is substituted for a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

[0076] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to achieve a beneficial or desired outcome (including a clinical outcome) when administered to a subject in need thereof, including a mammal (e.g., a human), and thus, an "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of treating sensorineural hearing loss or vestibular dysfunction, it is an amount of a composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that will correspond to such an amount will vary according to various factors, such as a given dose, pharmaceutical formulation, route of administration, type of disease or condition, subject (e.g., age, sex, weight) or identity of the host, etc., but can still be routinely determined by one skilled in the art. Likewise, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector disclosed herein is an amount that produces a beneficial or desired outcome in a subject compared to a control. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include an amount of each ingredient that would be effective when administered alone. As defined herein, a therapeutically effective amount of a composition, vector construct, or viral vector of the present disclosure can be readily determined by one of ordinary skill by conventional methods known in the art. The dosage regimen may be adjusted to provide the optimal therapeutic response.

[0077] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally present in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell, e.g., a human hair cell).

[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' cap formation, 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 "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that does not naturally occur in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell, e.g., a human hair cell). Exogenous materials include those materials that are provided to an organism by an external source or to (extracted from) a culture medium.

[0080] As used herein, the term "hair cell-specific expression" means that an RNA transcript or polypeptide is produced 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 versus non-hair cells) using any standard technique (e.g., quantitative RT PCR, immunohistochemistry, Western blot analysis, or measurement of fluorescence of a reporter gene (e.g., GFP) operably linked to a promoter). The hair cell-specific promoter induces expression (e.g., RNA or protein expression) of a transgene to which it is operably linked that is at least 50% greater (e.g., 50%, 75%, 100%, 125%, 150%, 175%, 200% or more greater) in hair cells than in 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 digit cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hansen cells, Claudius cells, inner sulcus cells, outer sulcus cells, spiral process 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.

[0081] As used herein, the terms "increase" and "decrease" refer to the regulation of a function, expression or activity that produces a greater or lesser amount, respectively, relative to a reference. For example, after the composition is administered as described herein, the amount of a marker of a measure (e.g., transgenic expression) as described herein may be increased or decreased in a subject 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 relative to the amount of the marker before administration. Typically, after administration, the metric is measured at the time when the effect has been achieved, for example, at least one week, one month, 3 months or 6 months after the start of the treatment regimen.

[0082] As used herein, the term "intron" refers to a region within the coding region of a gene whose nucleotide sequence is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of the RNA transcribed from the gene. Introns are transcribed into pre-mRNA, but are removed during processing and are not included in the mature mRNA.

[0083] As used herein, the term "linker" refers to a series of nucleotides that connect two different regions of a polynucleotide. The linker does not destroy the function of the two regions of the polynucleotide it connects.

[0084] As used herein, "topical" or "local administration" refers to administration to a specific part of the body intended for local rather than systemic effect. Examples of local administration are epidermal, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional administration, lymph node administration, intratumoral administration, administration to the inner ear, and administration to the mucous membranes of a subject, where the administration is intended to produce a local rather than systemic effect.

[0085] As used herein, the term "operably connected" refers to that a first molecule can be linked to a second molecule, wherein the arrangement of the molecule makes the first molecule affect the function of the second molecule. The term "operably connected" includes the juxtaposition of two or more parts (for example, a promoter and another sequence element) so that both parts function normally and allow at least one part to mediate the possibility of the function applied to at least one of the other parts. The two molecules may or may not be a part of a single adjacent molecule and may or may not be adjacent. For example, if the promoter regulates the transcription of the target transcribable polynucleotide molecule in the cell, the promoter is operably connected to the transcribable polynucleotide molecule. In other embodiments, if two parts of a transcriptional regulatory element are interconnected so that the transcriptional activation function of a part is not adversely affected by the presence of another part, the two parts are operably connected to each other. Two transcriptional regulatory elements can be operably connected to each other by means of a joint nucleic acid (for example, an intervening non-coding nucleic acid) or can be operably connected to each other in the absence of an intervening nucleotide.

[0086] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule to which another DNA segment can be connected. Plasmid is a carrier, a nucleic acid molecule capable of transporting another nucleic acid connected thereto. Some plasmids can replicate autonomously (e.g., bacterial plasmids and additional mammalian plasmids with bacterial replication origins) in the host cell into which they are introduced. Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. Some plasmids can direct the expression of the genes to which they are operably connected.

[0087] As used herein, the terms "nucleic acid" and "polynucleotide" used interchangeably herein refer to the polymeric form of nucleosides of any length. Generally, polynucleotides are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA connected by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application relates to polynucleotides, it should be understood that DNA, RNA are provided, and single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided in each case. As used herein, "polynucleotide sequence" may refer to polynucleotide material itself and / or biochemical characterization of sequence information of specific nucleic acids (i.e., a series of letters used as base abbreviations). Unless otherwise indicated, the polynucleotide sequences given herein are given in 5' to 3' directions.

[0088] As used herein, the term "complementarity" or "complementary" of a nucleic acid refers to a nucleotide sequence in one strand of a nucleic acid that forms hydrogen bonds with another sequence on the opposite nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are typically A to T and C to G. In RNA, they are typically C to G and U to A. Complementarity can be complete or substantially / fully complementary. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, wherein each base in the duplex is bound to a complementary base by Watson-Crick pairing. "Substantially" or "fully" complementary means that the sequence in one strand is not completely and / or fully complementary to the sequence in the opposite strand, but under a set of hybridization conditions (e.g., salt concentration and temperature), sufficient bonding occurs between the bases on the two strands to form a stable hybrid complex. Such conditions can be predicted by using the sequence and standard mathematical calculations to predict the Tm (melting temperature) of the hybridized strand, or by empirically determining the Tm using conventional methods. Tm includes the temperature at which 50% of the population of hybrid complexes formed between two nucleic acid chains is denatured (i.e., half of the population of double-stranded nucleic acid molecules is dissociated into single strands). At temperatures below Tm, the formation of hybrid complexes is favored, while at temperatures above Tm, the melting or separation of the chains in the hybrid complex is favored. The Tm of a nucleic acid with a known G+C content in a 1M NaCl aqueous solution can be estimated by using, for example, Tm=81.5+0.41 (%G+C), although other known Tm calculations take into account nucleic acid structural features.

[0089] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of a transgene.

[0090] " Percentage (%) of sequence identity " with respect to reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acid or amino acid identical with the nucleic acid or amino acid in reference polynucleotide or polypeptide sequence in candidate sequence after sequence alignment and introducing room when necessary to realize maximum sequence identity percentage.Comparison for determining nucleic acid or amino acid sequence identity percentage can be realized in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software.One skilled in the art can determine the parameters applicable to alignment sequence, including any algorithm required for maximum alignment in the full length of the compared sequence.For example, sequence comparison computer program BLAST can be used to generate sequence identity percentage value.As an illustration, given nucleic acid or amino acid sequence A for, with or for given nucleic acid or amino acid sequence B sequence identity percentage (it can be alternatively phrased as given nucleic acid or amino acid sequence A has for, with or for given nucleic acid or amino acid sequence B certain sequence identity percentage) is calculated as follows:

[0091] 100×(fraction X / Y)

[0092] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be understood that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0093] As used herein, the term "derivative" refers to a nucleic acid, peptide or protein or a variant or analog thereof that contains one or more mutations and / or chemical modifications compared to a corresponding full-length wild-type nucleic acid, peptide or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to a base moiety, a sugar moiety, a phosphate moiety, a phosphate-sugar backbone, or a combination thereof.

[0094] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject (such as a mammal, e.g., a human) in order to prevent, treat or control a particular disease or condition that affects or may affect the subject.

[0095] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms suitable for contact with a subject, such as a mammal (e.g., human) tissue without excessive toxicity, irritation, allergic response and other problematic complications commensurate with a reasonable benefit / risk ratio. Preferably, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals and more specifically humans.

[0096] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermal tissue), pancreatic juice, chorion, and cells).

[0097] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a target gene. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described in, for example, Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0098] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection, and the like.

[0099] As used herein, the terms "subject" and "patient" refer to animals (e.g., mammals, such as humans), veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of diseases (e.g., mice, rats). The subject to be treated according to the methods described herein can be a subject who has been diagnosed with hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance) or a subject at risk of developing these disorders. Diagnosis can be performed by any method or technique known in the art. Those skilled in the art will appreciate that the subject to be treated according to the present disclosure may have been subjected to standard testing, or may have been identified as a subject at risk without examination due to the presence of one or more risk factors associated with the disease or disorder.

[0100] As used herein, the term "transduction" and "transduce" refer to a method of introducing a vector construct or a portion thereof into a cell. Where the vector construct is contained in a viral vector such as an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or a portion thereof into the cell genome.

[0101] As used herein, "treatment" and "treating" of a condition, disorder, or illness may include: (1) preventing, delaying, or reducing the incidence and / or likelihood of occurrence of at least one clinical or subclinical symptom of a condition, disorder, or illness that develops in a subject who may have or is susceptible to the condition, disorder, or illness but does not yet experience or exhibit clinical or subclinical symptoms of the condition, disorder, or illness; or (2) inhibiting the condition, disorder, or illness, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the condition, disorder, or illness or at least one clinical or subclinical symptom thereof. The benefit to the subject to be treated is statistically significant or at least perceptible to the patient or physician.

[0102] As used herein, the term "vector" includes nucleic acid vectors, for example, DNA vectors, such as plasmids, cosmids or artificial chromosomes, RNA vectors, viruses or any other suitable replicons (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 in, for example, 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, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express transgenics as described herein include vectors containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other available vectors for expressing transgenics contain polynucleotide sequences that increase the translation rate of the transgenic or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseoin.

[0103] As used herein, the term "vestibular hair cells" refers to a group of specialized cells in the inner ear that are involved in sensing movement and contribute to the sense of balance and spatial orientation. Vestibular hair cells are located in the semicircular canals and otoliths of the inner ear. Damage to vestibular hair cells and genetic mutations that disrupt vestibular hair cell function are associated with vestibular dysfunction (such as vertigo and disequilibrium).

[0104] As used herein, the term "wild type" refers to the genotype with the highest frequency for a particular gene in a given organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1A-1B The AAV vector was constructed to express GFP under the control of the cytomegalovirus (CMV) promoter. Figure 1A ) or in the murine Myo15 promoter (SEQ ID NO: 13, Figure 1BA series of fluorescent images of the mouse cochlea transduced with an AAV vector expressing GFP under the control of ). The AAV-Myo15-GFP virus was infused into 6-8 week old C57Bl / 6J male mice through the posterior semicircular canal. The mice were allowed to recover from surgery and euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bones were harvested and decalcified in 8% EDTA for 3 days. The cochlea was dissected from the decalcified temporal bones and mounted on a slide for confocal imaging. Using a ubiquitous promoter, AAV-CMV-GFP induces GFP expression in many cell types within the cochlea, including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia ( Figure 1A Using a hair cell-specific promoter, AAV-Myo15-GFP was induced to express only in inner and outer hair cells ( Figure 1B ).

[0106] Figure 2 is used in the murine Myo15 promoter (SEQ ID NO: 13, Figure 2 ) of the vestibular system regions (utricle, saccule, posterior crista (PC), anteriorcrista (AC), and horizontal crista (HC)) of mice transduced with an AAV vector expressing GFP under the control of AAV-Myo15. AAV-Myo15-GFP virus was infused into 6-8 week old C57Bl / 6J male mice through the posterior semicircular canal. The mice were allowed to recover from surgery and euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bones were harvested and decalcified in 8% EDTA for 3 days. The vestibular organs were dissected from the decalcified temporal bones and mounted on slides for imaging. Using a hair cell-specific promoter, AAV-Myo15-GFP induces expression only in vestibular hair cells ( Figure 2 ).

[0107] Figure 3A-3B is used in the murine Myo151 kb promoter (SEQ ID NO: 15, Figure 3A ) or Myo15 mini-intron promoter (SEQ ID NO: 16, Figure 3B Serial fluorescence images of mouse cochlea transduced with AAV 2 / 9 viral vector expressing GFP under the control of ). Figure 3A-3B As shown, both the murine Myo15 1 kb (SEQ ID NO: 15) and murine Myo15 mini-intron (SEQ ID NO: 16) promoters induced the expression of GFP specifically in hair cells.

[0108] Figures 4A-4GA series of mouse cochlear images showing the specificity of the murine and human Myo15 promoters. Neonatal mouse cochlear explants were incubated with a 50-kb genomic DNA sequence containing the 0.6 kb human Myo15 promoter (SEQ ID NO: 26; Figure 4A )、1.2 kb human Myo15 promoter (SEQ ID NO: 25; Figure 4B ) or 1 kb mouse Myo15 promoter (SEQ ID NO: 15; Figure 4C ) were treated in culture with an AAV1 vector encoding GFP under the control of GFP (scale bar = 100 μm). GFP signal as detected with anti-GFP antibody is shown. In separate experiments, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb murine Myo15-GFP was delivered to the cochlea of ​​adult mice by round window injection. Whole mount images ( Figure 4D-4E ) or slice ( Figure 4F-4G ) shows hair cell-specific signals in the apex, middle, and base of the cochlea, as detected with anti-GFP antibody. Figure 4F The arrows in the figure point to GFP-labeled cochlear hair cells. Figure 4F and 4G As shown, expression in the case of the 1.2 kb human Myo15 promoter and the 1 kb mouse Myo15 promoter is primarily restricted to inner hair cells.

[0109] Figures 5A-5C is a series of fluorescent images of non-human primate cochleae showing that the Myo15 promoter restricts GFP expression to hair cells in the non-human primate cochleae. Figure 5A is a confocal image of the cochlea from a non-human primate that received a local injection of AAV1-CMV-GFP through the round window membrane. Tissue was harvested 28 days after injection. Native GFP fluorescence is shown. GFP expression was detected in a wide variety of cell types throughout the organ. Figure 5B is from non-human primates injected with AAV1-Myo15 (SEQ ID NO: 13)-GFP and Figure 5A Confocal image of a cochlea processed in the same manner as the cochlea in Figure 1. GFP expression is confined to hair cells. Figure 5C yes Figure 5B Magnified view of the hair cells in the boxed area shown.

[0110] Figure 61.6kb Myo15 promoter (SEQ ID NO: 13) enhances the biological efficacy of AAV-mouse TMC1 vector in Tmc1 knockout (KO) mice compared to the ubiquitous CMV promoter. Tmc1 KO mice were injected at postnatal day 2 (P2) and auditory brainstem responses (ABRs) were assessed at the indicated ages. ABR thresholds were plotted as a function of stimulation frequency for homozygous (light gray line with open circles) and heterozygous (dark gray line through black circles) Tmc1 KO mice initially and homozygous Tmc1 KO mice injected with AAV-CMV-mouse TMC1 (CMV_mTmc1; dark gray line with open circles) or AAV-Myo15 (SEQ ID NO: 13)-mouse TMC1 (PdBx_mTmc1; P23, light gray line through light gray circles; P28, dark gray line with solid circles). The recovery of ABR thresholds was greatly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 compared with AAV-CMV-mouse TMC1.

[0111] Figure 7A-7B is a series of fluorescent images of the inner ear of cynomolgus monkeys treated with an AAV1 vector expressing GFP under the control of the 1.2 kb human myosin 15 promoter (SEQ ID NO: 25). Bilateral (n = 1 animal) injections were used to deliver 30 μL of AAV1-1.2 kb myosin 15-GFP (virus titer 1.06×10 13 genomic copies / mL) were administered to the round window of the inner ear. Four weeks after injection, the inner ear was removed and the surface of the basement membrane was prepared. GFP expression was used to monitor myosin 15 promoter activity ( Fig. 7A , one of the representative ears is shown). AAV1-1.2 kb myosin 15-GFP transduction of the cynomolgus monkey inner ear resulted in GFP expression throughout the basal-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex ( Fig. 7A , frequency in kHz). High-magnification images at 2.8 kHz show that GFP expression was observed in inner hair cells (IHC) and outer hair cells (OHC) ( Figure 7B , upper panel). Immunohistochemistry of Myo7A was used to visualize hair cells, and cell nuclei were stained with DAPI ( Figure 7B , middle and bottom panels).

[0112] Figures 8A-8Bis a series of fluorescent images of the inner ear of cynomolgus monkeys treated with an AAV1 vector expressing GFP under the control of the 0.6 kb human myosin 15 promoter (SEQ ID NO: 26). Bilateral (n = 1 animal) injections were used to deliver 30 μL of AAV1-0.6 kb myosin 15-GFP (virus titer 1.22×10 13 genomic copies / mL) were administered to the round window of the inner ear. Four weeks after injection, the inner ear was removed and the surface of the basement membrane was prepared. GFP expression was used to monitor myosin 15 promoter activity ( Fig. 8A , one of the representative ears is shown). AAV1-0.6 kb myosin 15-GFP transduction of the cynomolgus monkey inner ear resulted in GFP expression throughout the basal-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex ( Fig. 8A , frequency in kHz). High-magnification images at 2.8 kHz show that GFP expression was observed in inner hair cells (IHC) and outer hair cells (OHC) ( Figure 8B , upper panel). Immunohistochemistry of Myo7A was used to visualize hair cells, and cell nuclei were stained with DAPI ( Figure 8B , middle and bottom panels).

[0113] Fig. 9 is a bar graph showing quantification of GFP expression from three different murine Myo15 (mMyo15) promoters with hair cell specific expression using quantitative PCR (qPCR). Transcription levels were normalized to the hair cell specific gene Myo7a. Expression levels of GFP reporters from the mMyo15 1.6 kb (SEQ ID NO: 13), 1 kb (SEQ ID NO: 15), and 0.5 kb (SEQ ID NO: 16) promoters were comparable to each other and robust.

[0114] Fig.10 Graph showing the results of quantitative image analysis of GFP fluorescence in myosin 7a (Myo7a)-positive hair cells driven by a variety of different promoters, including truncated chimeric CMV-chicken β-actin (smCBA) promoter, 1.6 kb mMyo15 promoter (SEQ ID NO: 13), 1 kb mMyo15 promoter (SEQ ID NO: 15), 0.5 kb mMyo15 promoter (SEQ ID NO: 16), 1.2 kb hMyo15 promoter (SEQ ID NO: 25) and 0.6 kb hMyo15 promoter (SEQ ID NO: 26). The GFP protein expression level per cell indicates the difference in expression between the Myo15 promoters, with the mMyo15 1 kb promoter producing the highest level of GFP protein per cell.

[0115] Figures 11A-11J is a series of whole mount fluorescence images of neonatal cochlear explant cultures infected with viral vectors containing Myo15 promoter variants of SEQ ID NOs: 16 and 27-35. The promoter sequence of SEQ ID NO: 27 (Myo15 A) is neither sufficient nor necessary for general promoter activity or hair cell specificity, as Fig. 11B (SEQ ID NO: 27), 11E (SEQ ID NO: 30), 11F (SEQ ID NO: 31) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 27 does provide a splice donor site, which increases the expression level when paired with a splice acceptor site, such as Fig.11A (SEQ ID NO: 16) and 11H (SEQ ID NO: 33), thereby introducing an intron into the promoter sequence. The promoter sequence of SEQ ID NO: 28 (Myo15 B) alone was found to be a core promoter that confers general promoter activity, such as Fig. 11C (SEQ ID NO:28), but does not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO:29 (Myo15C), as shown in Fig. 11C (SEQ ID NO: 29) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 29 (Myo15 C) was found to be an enhancer that restricts the activity of the core promoter (SEQ ID NO: 28) to inner hair cells and outer hair cells, as shown in FIG. Fig.11G (SEQ ID NO: 32), but is not sufficient for general promoter activity in the absence of the core promoter, such as Fig.11D (SEQ ID NO: 29). The promoter sequences of SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 were reordered into various combinations and produced to result in hair cell-specific promoter activity, such as Fig.11H (SEQ ID NO:33) and 11I (SEQ ID NO:34), although not all rearrangements are tolerated, such as Fig.11J (shown in SEQ ID NO:35).

[0116] Fig.12is a bar graph showing quantification of GFP expression from promoter variants with hair cell specific expression as measured by qPCR. Transcript levels were normalized to the hair cell specific gene Myo7a. Expression levels of GFP reporter genes from Myo15 0.5 kb (SEQ ID NO: 16), Myo15B-C (SEQ ID NO: 32), and Myo15 BAC (SEQ ID NO: 33) promoters were comparable to each other and robust. The Myo15 CAB promoter (SEQ ID NO: 34) was generated by rearranging the positions of the splice donor and acceptor sites, resulting in cryptic splicing to the middle of the GFP cDNA, as evidenced by higher expression levels detected with a GFP probe located in the 3' end of the transcript compared to the 5' end or the middle of the transcript.

[0117] Figures 13A-13B is a series of graphs showing quantitative image analysis of GFP expression levels in Myo7a-positive hair cells. The expression level of GFP protein per cell shows differences in expression between Myo15 promoters ( Figures 13A-13B ), including the 0.5 kb mMyo15 promoter (also known as the Myo15 ABC promoter; SEQ ID NO: 16), the Myo15 AB promoter (SEQ ID NO: 30), the Myo15 AC promoter (SEQ ID NO: 31), the Myo15B-C promoter (SEQ ID NO: 32), the Myo15 A promoter (SEQ ID NO: 27), the Myo15 B promoter (SEQ ID NO: 28), the Myo15 C promoter (SEQ ID NO: 29), the Myo15 BAC promoter (SEQ ID NO: 33), the Myo15 CAB promoter (SEQ ID NO: 34), and the Myo15B-CA promoter (SEQ ID NO: 35). The Myo15 ABC promoter (SEQ ID NO: 16) produced the highest GFP expression per cell ( Fig.13A ). Based on tissue autofluorescence (for Fig.13A , <2000 units and for Fig. 13B , <6000 units) to determine the background fluorescence level for each data set. DETAILED DESCRIPTION

[0118] Compositions and methods for inducing transgene expression specifically in hair cells (e.g., cochlear and / or vestibular hair cells) are described herein. The invention features polynucleotides containing a region of the myosin 15 (Myo15) promoter that are capable of expressing transgenes specifically in cochlear hair cells. The invention also features nucleic acid vectors containing these promoters operably linked to polynucleotides encoding polypeptides. The compositions and methods described herein can be used to specifically express polynucleotides encoding hair cell proteins in cochlear and vestibular hair cells, and thus, the compositions described herein can be administered to a subject (such as a mammalian subject, e.g., a human) to treat conditions caused by dysfunction of hair cells, such as hearing loss or vestibular dysfunction.

[0119] Hair cells

[0120] Hair cells are sensory cells of the auditory and vestibular systems residing in the inner ear. Cochlear hair cells are sensory cells of the auditory system and consist of two main cell types: inner hair cells responsible for sensing sound and outer hair cells that are thought to amplify low-volume sounds. Vestibular hair cells are located in the semicircular canals and otolith organs of the inner ear and are involved in the sense of movement, which contributes to the sense of balance and spatial orientation. Hair cells are named after the stereocilia that protrude from the top surface of the cells to form a hair cell bundle. Deflection of the stereocilia (e.g., by sound waves in the cochlear hair cells, or by rotational or linear acceleration in the vestibular hair cells) causes mechanically gated ion channels to open, allowing the hair cells to release neurotransmitters to activate nerves, thereby converting mechanical sound or motion signals into electrical signals that can be transmitted to the brain. Cochlear hair cells are essential for normal hearing, and damage to cochlear hair cells and genetic mutations that destroy cochlear hair cell function are associated with hearing loss and deafness. Damage to vestibular hair cells and genetic mutations that disrupt vestibular hair cell function are associated with vestibular dysfunction, such as loss of balance and vertigo (e.g., dizziness). Gene therapy has recently become an attractive treatment for hearing loss and vestibular dysfunction; however, the art lacks methods for specifically targeting nucleic acid vectors used in gene therapy to hair cells.

[0121] Myosin 15

[0122] Myo15 is an unconventional actin-based molecular motor that regulates the development of stereocilia. Mice carrying mutations in Myo15 have been found to have short stereocilia and severe hearing loss and vestibular dysfunction, and mutations in the human ortholog Myo15A cause non-syndromic autosomal recessive deafness DFNB3. Myo15 has been observed to be localized to stereocilia and is essential for the development and maintenance of stereocilia. The localization pattern suggests that Myo15 may be specifically expressed in hair cells. However, the Myo15 promoter has not been isolated and characterized previously. We identified evolutionarily conserved blocks of regulatory elements that may constitute the promoter in the orthologous genomic sequences and found that they are located more than 7200 base pairs (bp) upstream of the translation start site. This genomic region is too large to be used in combination with adeno-associated virus (AAV) vectors with a maximum packaging capacity of 4.7 kb to deliver target transgenes for gene therapy.

[0123] The present invention is based in part on the discovery of a region upstream of the Myo15 translation start site that can be used to promote specific expression of transgenes in hair cells (e.g., cochlear and / or vestibular hair cells). Therefore, the compositions and methods described herein can be used to express target genes (e.g., genes related to hair cell development, function, cell fate determination, regeneration, survival or maintenance, or genes known to be disrupted (e.g., mutated) in subjects with hearing loss or vestibular dysfunction) in hair cells to treat subjects with or at risk of hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness or loss of balance).

[0124] Mouse myosin 15 promoter

[0125] The polynucleotides of the compositions and methods described herein comprise nucleic acid sequences from regions of the murine Myo15 locus capable of specifically expressing a transgene in hair cells, or variants thereof, such as nucleic acid sequences having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to regions of the murine Myo15 locus capable of specifically expressing a transgene in hair cells. These regions include nucleic acid sequences immediately preceding the murine Myo15 translation start site and upstream regulatory elements located more than 5 kb from the murine Myo15 translation start site. The polynucleotides of the compositions and methods described herein may optionally comprise a linker operably linked to the region of the murine Myo15 locus capable of specifically expressing a transgene in hair cells, or the region of the murine Myo15 locus may be directly linked without an intervening linker.

[0126] In some embodiments, the polynucleotides described herein contain a first region (upstream regulatory element) linked (e.g., operably linked) to a second region, wherein the first region has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with a region comprising the first non-coding exon of the Myo15 gene (a nucleic acid from -6755 to -7209 relative to the translation start site of murine Myo15, the sequence of which is shown in SEQ ID NO: 1) or a functional part or derivative thereof, and the second region has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with a nucleic acid sequence immediately preceding the translation start site of murine Myo15 (a nucleic acid from -1 to -1157 relative to the translation start site of murine Myo15, the sequence of which is shown in SEQ ID NO: 2) or a functional part or derivative thereof. The functional portion of SEQ ID NO:1 may have a nucleic acid sequence from -7166 to -7091 relative to the murine Myo15 translation start site (shown in SEQ ID NO:3) and / or a nucleic acid sequence from -7077 to -6983 relative to the murine Myo15 translation start site (shown in SEQ ID NO:4). The first region may contain the nucleic acid sequence of SEQ ID NO:3 fused to the nucleic acid sequence of SEQ ID NO:4 without using an intervening nucleic acid, as shown in SEQ ID NO:5, or the first region may contain the nucleic acid sequence of SEQ ID NO:4 fused to the nucleic acid sequence of SEQ ID NO:3 without using an intervening nucleic acid, as shown in SEQ ID NO:6. Alternatively, the first region may contain the sequences of SEQ ID NO:3 and SEQ ID NO:4 connected by an endogenous intervening nucleic acid sequence (e.g., the first region may have or include a nucleic acid sequence from -7166 to -6983 relative to the murine Myo15 translation start site, as shown in SEQ ID NO:7 and SEQ ID NO:27) or a nucleic acid linker. In a polynucleotide in which the first region contains both SEQ ID NO:3 and SEQ ID NO:4, the two sequences can be included in any order (e.g., SEQ ID NO:3 can be linked to (e.g., in front of) SEQ ID NO:4, or SEQ ID NO:4 can be linked to (e.g., in front of) SEQ ID NO:3). The functional portion of SEQ ID NO:2 can have a nucleic acid sequence from -590 to -509 relative to the Myo15 translation start site (shown in SEQ ID NO:8) and / or a nucleic acid sequence from -266 to -161 relative to the murine Myo15 translation start site (shown in SEQ ID NO:9).In some embodiments, the sequence containing SEQ ID NO: 8 has the sequence of SEQ ID NO: 28. In some embodiments, the sequence containing SEQ ID NO: 9 has the sequence of SEQ ID NO: 29. The second region may contain the nucleic acid sequence of SEQ ID NO: 8 fused to the nucleic acid sequence of SEQ ID NO: 9 without an intervening nucleic acid, as shown in SEQ ID NO: 10, or the second region may contain the nucleic acid sequence of SEQ ID NO: 9 fused to the nucleic acid sequence of SEQ ID NO: 8 without an intervening nucleic acid, as shown in SEQ ID NO: 11. The second region may contain the nucleic acid sequence of SEQ ID NO: 28 fused to the nucleic acid sequence of SEQ ID NO: 29 without an intervening nucleic acid, as shown in SEQ ID NO: 32, or the second region may contain the nucleic acid sequence of SEQ ID NO: 29 fused to the nucleic acid sequence of SEQ ID NO: 28 without an intervening nucleic acid. Alternatively, the second region may contain the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 connected by an endogenous intervening nucleic acid sequence (e.g., the second region may have a nucleic acid sequence from -590 to -161 relative to the murine Myo15 translation start site, as shown in SEQ ID NO: 12) or a nucleic acid linker. In a polynucleotide in which the second region contains both SEQ ID NO: 8 and SEQ ID NO: 9, the two sequences may be included in any order (e.g., SEQ ID NO: 8 may be connected to (e.g., in front of) SEQ ID NO: 9, or SEQ ID NO: 9 may be connected to (e.g., in front of) SEQ ID NO: 8).

[0127] The first region and the second region of the polynucleotide can be directly connected or can be connected by a nucleic acid joint. For example, the polynucleotide may contain a sequence of SEQ ID NO:1 or its functional part or derivative (e.g., any one or more of SEQ ID NO:8-12, 28, 29 and 32, such as SEQ ID NO 8 and 9) without using an intervening nucleic acid fusion to SEQ ID NO:2 or its functional part or derivative (e.g., any one or more of SEQ ID NO:3-7 and 27, such as SEQ ID NO 3 and 4). For example, the nucleic acid sequence of the polynucleotide produced by SEQ ID NO:1 directly fused to SEQ ID NO:2 is shown in SEQ ID NO:13. Alternatively, a joint can be used to connect the sequence of SEQ ID NO:1 or its functional part or derivative (e.g., any one or more of SEQ ID NO:3-7 and 27, such as SEQ ID NO 3 and 4) to SEQ ID NO:2 or its functional part or derivative (e.g., any one or more of SEQ ID NO:8-12, 28, 29 and 32, such as SEQ ID NO 8 and 9). Exemplary polynucleotides containing functional portions of both SEQ ID NO: 1 and SEQ ID NO: 2 are provided in SEQ ID NO: 15, 16, 30 and 31.

[0128] The length of the nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp or less). The nucleic acid linker that can be used in the polynucleotides described herein does not disrupt the ability of the polynucleotides of the invention to induce transgene expression in hair cells.

[0129] In some embodiments, the sequence of SEQ ID NO: 1 or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, such as SEQ ID NOs 3 and 4) is linked (e.g., operably linked) to the sequence of SEQ ID NO: 2 or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29 and 32, such as SEQ ID NOs 8 and 9), and, in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 2 or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29 and 32, such as SEQ ID NOs 8 and 9) is linked (e.g., operably linked) to the sequence of SEQ ID NO: 1 or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, such as SEQ ID NOs 3 and 4)). For example, the nucleic acid sequence of the polynucleotide generated by direct fusion of SEQ ID NO: 2 to SEQ ID NO: 1 is shown in SEQ ID NO: 14. An example of a polynucleotide in which the functional part or derivative of SEQ ID NO: 2 precedes the functional part or derivative of SEQ ID NO: 1 is provided in SEQ ID NO: 35. Regardless of the order, as described above, the sequence of SEQ ID NO: 1 or a functional part or derivative thereof and the sequence of SEQ ID NO: 2 or a functional part or derivative thereof can be linked by direct fusion or a nucleic acid linker.

[0130] In some embodiments, the polynucleotides described herein comprise a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acid from -6755 to -7209 relative to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 1) or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 1 may have a nucleic acid sequence from -7166 to -7091 relative to the murine Myo15 translation start site (set forth in SEQ ID NO: 3) and / or a nucleic acid sequence from -7077 to -6983 relative to the Myo15 translation start site (set forth in SEQ ID NO: 4). The polynucleotide may contain the nucleic acid sequence of SEQ ID NO:3 fused to the nucleic acid sequence of SEQ ID NO:4 without using an intervening nucleic acid, as shown in SEQ ID NO:5, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO:4 fused to the nucleic acid of SEQ ID NO:3 without using an intervening nucleic acid, as shown in SEQ ID NO:6. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO:3 and SEQ ID NO:4 connected by an endogenous intervening nucleic acid sequence (e.g., the first region may have or include a nucleic acid sequence from -7166 to -6983 relative to the murine Myo15 translation start site, as shown in SEQ ID NO:7 and SEQ ID NO:27) or a nucleic acid linker. In a polynucleotide containing both SEQ ID NO:3 and SEQ ID NO:4, the two sequences may be included in any order (e.g., SEQ ID NO:3 may be connected to (e.g., in front of) SEQ ID NO:4, or SEQ ID NO:4 may be connected to (e.g., in front of) SEQ ID NO:3).

[0131] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to a nucleic acid sequence immediately upstream of the murine Myo15 translation start site (nucleic acid from -1 to -1157 relative to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 2) or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 2 may have a nucleic acid sequence from -590 to -509 relative to the murine Myo15 translation start site (set forth in SEQ ID NO: 8) and / or a nucleic acid sequence from -266 to -161 relative to the murine Myo15 translation start site (set forth in SEQ ID NO: 9). In some embodiments, the sequence containing SEQ ID NO: 8 has a sequence of SEQ ID NO: 28. In some embodiments, the sequence containing SEQ ID NO: 9 has a sequence of SEQ ID NO: 29. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 8 fused to the nucleic acid sequence of SEQ ID NO: 9 without an intervening nucleic acid, as shown in SEQ ID NO: 10, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 9 fused to the nucleic acid sequence of SEQ ID NO: 8 without an intervening nucleic acid, as shown in SEQ ID NO: 11. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 28 fused to the nucleic acid sequence of SEQ ID NO: 29 without an intervening nucleic acid, as shown in SEQ ID NO: 32, or the second region may contain the nucleic acid sequence of SEQ ID NO: 29 fused to the nucleic acid sequence of SEQ ID NO: 28 without an intervening nucleic acid. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 connected by an endogenous intervening nucleic acid sequence (e.g., the second region may have a nucleic acid sequence from -590 to -161 relative to the murine Myo15 translation start site, as shown in SEQ ID NO: 12) or a nucleic acid linker. In a polynucleotide containing both SEQ ID NO:8 and SEQ ID NO:9, the two sequences can be included in any order (e.g., SEQ ID NO:8 can be linked to (e.g., preceded by) SEQ ID NO:9, or SEQ ID NO:9 can be linked to (e.g., preceded by) SEQ ID NO:8).

[0132] In some embodiments, the polynucleotides described herein comprise a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to a region containing the first noncoding exon of a Myo15 gene (a nucleic acid from -6755 to -7209 relative to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 1), wherein the region containing the first noncoding exon of a Myo15 gene is flanked on either side by a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to a nucleic acid sequence immediately upstream of the murine Myo15 translation start site (a nucleic acid from -1 to -1157 relative to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 2). For example, a functional portion or derivative of SEQ ID NO: 2, such as SEQ ID NO: 8 or 28, can be directly fused or linked to a portion of SEQ ID NO: 1, such as any one of SEQ ID NOs: 3-7 and 27, through a nucleic acid linker, which is directly fused or linked to a different functional portion of SEQ ID NO: 2, such as SEQ ID NO: 9 or 29. In other embodiments, a functional portion or derivative of SEQ ID NO: 2, such as SEQ ID NO: 9 or 29, can be directly fused or linked to a portion of SEQ ID NO: 1, such as any one of SEQ ID NOs: 3-7 and 27, through a nucleic acid linker, which is directly fused or linked to a different functional portion of SEQ ID NO: 2, such as SEQ ID NO: 8 or 28. For example, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the nucleic acid sequences of SEQ ID NOs:28, 27, and 29 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the nucleic acid sequence of SEQ ID NO:33. In some embodiments, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the nucleic acid sequences of SEQ ID NOs:29, 27, and 28 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the nucleic acid sequence of SEQ ID NO:34.

[0133] Human myosin 15 promoter

[0134] The polynucleotides of the compositions and methods described herein may also include a nucleic acid sequence from a region of the human Myo15 locus capable of specifically expressing a transgene in hair cells, or a variant thereof, such as a nucleic acid sequence having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with a region of the human Myo15 locus capable of specifically expressing a transgene in hair cells. The polynucleotides of the compositions and methods described herein may optionally include a linker operably linked to a region of the human Myo15 locus capable of specifically expressing a transgene in hair cells, or the region of the human Myo15 locus may be directly linked without an intervening linker.

[0135] In some embodiments, the polynucleotides described herein contain a first region linked (e.g., operably linked) to a second region, the first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the sequence shown in SEQ ID NO: 17 or a functional portion or derivative thereof, and the second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with the sequence shown in SEQ ID NO: 18 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 17 may have the sequence shown in SEQ ID NO: 19. The functional portion of SEQ ID NO: 18 may have the sequence shown in SEQ ID NO: 20 and / or the sequence shown in SEQ ID NO: 21. The second region may contain the nucleic acid sequence of SEQ ID NO: 20 fused to the nucleic acid sequence of SEQ ID NO: 21 without using an intervening nucleic acid, as shown in SEQ ID NO: 22, or the second region may contain the nucleic acid sequence of SEQ ID NO: 21 fused to the nucleic acid sequence of SEQ ID NO: 20 without using an intervening nucleic acid, as shown in SEQ ID NO: 23. Alternatively, the second region may contain the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 connected by an endogenous intervening nucleic acid sequence (as shown in SEQ ID NO: 24) or a nucleic acid linker. In a polynucleotide in which the second region contains both SEQ ID NO: 20 and SEQ ID NO: 21, the two sequences may be included in any order (e.g., SEQ ID NO: 20 may be connected to (e.g., in front of) SEQ ID NO: 21, or SEQ ID NO: 21 may be connected to (e.g., in front of) SEQ ID NO: 20).

[0136] The first region and the second region of the polynucleotide may be directly connected or may be connected by a nucleic acid joint. For example, the polynucleotide may contain a sequence of SEQ ID NO:17 or a functional part or derivative thereof (e.g., SEQ ID NO:19) that is not fused to a sequence of SEQ ID NO:18 or a functional part thereof or derivative thereof (e.g., SEQ ID NO:20-24, any one or more, such as SEQ ID NO:20 and / or 21) without using an intervening nucleic acid. Alternatively, a joint may be used to connect the sequence of SEQ ID NO:17 or a functional part thereof or derivative thereof (e.g., SEQ ID NO:19) to the sequence of SEQ ID NO:18 or a functional part thereof or derivative thereof (e.g., SEQ ID NO:20-24, any one or more, such as SEQ ID NO:20 and / or 21). Exemplary polynucleotides containing the functional parts of both SEQ ID NO:17 and SEQ ID NO:18 are provided in SEQ ID NO:25 and 26.

[0137] In some embodiments, the sequence of SEQ ID NO: 17, or a functional part or derivative thereof (e.g., SEQ ID NO: 19) is linked (e.g., operably linked) to the sequence of SEQ ID NO: 18, or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and 21), and, in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 18, or a functional part or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and / or 21) is linked (e.g., operably linked) to the sequence of SEQ ID NO: 17, or a functional part or derivative thereof (e.g., SEQ ID NO: 19). Regardless of the order, as described above, the sequence of SEQ ID NO: 17, or a functional part or derivative thereof, and the sequence of SEQ ID NO: 18, or a functional part or derivative thereof, can be linked by direct fusion or a nucleic acid linker.

[0138] In some embodiments, the polynucleotides described herein comprise a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to a region containing the sequence shown in SEQ ID NO: 17 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 17 may have the nucleic acid sequence shown in SEQ ID NO: 19.

[0139] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the sequence shown in SEQ ID NO: 18 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 18 may have the sequence shown in SEQ ID NO: 20 and / or the sequence shown in SEQ ID NO: 21. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 20 fused to the nucleic acid sequence of SEQ ID NO: 21 without using an intervening nucleic acid, as shown in SEQ ID NO: 22, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 21 fused to the nucleic acid sequence of SEQ ID NO: 20 without using an intervening nucleic acid, as shown in SEQ ID NO: 23. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 connected by an endogenous intervening nucleic acid sequence (e.g., as shown in SEQ ID NO: 24) or a nucleic acid linker. In a polynucleotide containing both SEQ ID NO:20 and SEQ ID NO:21, the two sequences can be included in any order (e.g., SEQ ID NO:20 can be linked to (e.g., preceded by) SEQ ID NO:21, or SEQ ID NO:21 can be linked to (e.g., preceded by) SEQ ID NO:20).

[0140] The length of the nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp or less). The nucleic acid linker that can be used in the polynucleotides described herein does not disrupt the ability of the polynucleotides of the invention to induce transgene expression in hair cells.

[0141] The aforementioned nucleic acid sequences are summarized in Table 2 below.

[0142] Table 2: Exemplary nucleotide sequences for use in the polynucleotides described herein

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Additional polynucleotides that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the nucleic acid sequences listed in Table 2, and functional portions or derivatives of the nucleic acid sequences listed in Table 2.

[0162] The aforementioned polynucleotides may be included in a nucleic acid vector and operably linked to a transgene to specifically express the transgene in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). In some embodiments, the transgene encodes a protein related to hair cell function, hair cell development, hair cell fate determination, hair cell regeneration, hair cell survival, or hair cell maintenance, or the transgene is a wild-type form of a gene that has been found to be mutated in subjects with hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction (e.g., vertigo, dizziness, or loss of balance). According to the methods described herein, a composition containing one or more of the aforementioned polynucleotides (e.g., one or more polynucleotides listed in Table 2) may be administered to a subject, the polynucleotide being operably linked to a transgene encoding a therapeutic protein for treating hearing loss and / or vestibular dysfunction. In some embodiments,The transgene encodes a protein selected from the group consisting of: Actin gamma 1 (ACTG1), Fascin actin bundling protein 2, Retinal (FSCN2), Radixin (RDX), POU class 4 homeobox 3 (POU4F3), TRIO and F-actin binding protein (TRIOBP), Taperin (TPRN), Xin actin binding repeat containing 2 (XIRP2), Atonal BHLH transcription factor 1 (ATOH1), Growth factor independent 1 transcription repressor (GFI1), Cholinergic receptor nicotinic acid alpha 9 subunit (CHRNA9), Calcium and integrin binding family member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), Otoferlin (OTOF), MKRN2 opposite strand (MKRN2OS), LIM homeobox protein 3 (LHX3) , transmembrane channel-like protein 1 (TMC1), myosin 15 (MYO15), myosin 7A (MYO7A), myosin 6 (MYO6), myosin IIIA (MYO3A), myosin IIIB (MYO3B), glutaredoxin domain-containing cysteine-rich protein 1 (GRXCR1), protein tyrosine phosphatase receptor type Q (PTPRQ), late cornification envelope protein 6A (LCE6A), lipoxygenase homology domain-containing protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATP ATP2B2, calcium and integrin binding family member 2 (CIB2), calcium voltage-gated channel auxiliary subunit α2δ4 (CACNA2D4), calcium binding protein 2 (CABP2), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like protein 2 (EPS8L2), Espin (ESPN), Espin-like protein (ESPNL), peripherin 2 (PRPH2), stiff ciliary protein (STRC), solute carrier family 8 member A2 (SLC8A2), CCHC-type zinc finger-containing Protein 12 (ZCCHC12), proteins containing leucine-rich transmembrane and O-methyltransferase domains (LRTOMT2, LRTOMT1), USH1 protein network component Harmonin (USH1C), protein containing extracellular leucine-rich repeats and fibronectin type III domains 1 (ELFN1), tetratricopeptide repeat protein 24 (TTC24), Dystrotelin (DYTN), Kielin / tendin-like protein (KCP), Coiled-coil Glutamate Rich Protein 2,CCER2), Leucine-rich Repeat and Transmembrane Domain-containing protein 2 (LRTM2), KCNA10, NTF3, CLRN1, CLRN2, SKOR1, Tctex1 domain-containing protein 1 (TCTEX1D1), Fc receptor-like B (FCRLB), SLC17A8, glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2), BDNF, SERPINE3, and Nescient Helix-loop Helix 1 (Nescient Helix-loop Helix 1). 1, NHLH1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), transcription activator 6 (ATF6), eukaryotic translation initiation factor 2α kinase 3 (PERK), serine / threonine-protein kinase / endoribonuclease IRE1 (IRE1) and binding immunoglobulin protein (BIP).

[0163] Expression of foreign nucleic acids in mammalian cells

[0164] Mutations in various genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, have been associated with sensorineural hearing loss, and some of these mutations, such as mutations in MYO7A, are also associated with vestibular dysfunction. The compositions and methods described herein can be used to induce or increase the specific expression of a protein encoded by a target gene (e.g., a wild-type form of a gene associated with hearing loss and / or vestibular dysfunction, or a gene involved in hair cell development, function, cell fate determination, regeneration, survival or maintenance) in hair cells (e.g., cochlear and / or vestibular hair cells) by administering a nucleic acid vector comprising a Myo15 promoter operably linked to a nucleic acid sequence encoding a target protein (e.g., a polynucleotide comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1 or a functional part or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2 or a functional part or derivative thereof, the polynucleotide optionally comprising a linker connecting the first region and the second region). Extensive methods have been established to deliver proteins to mammalian cells and to stably express protein-encoding genes in mammalian cells.

[0165] Proteins that can be expressed in conjunction with the compositions described herein (e.g., when a protein-encoding transgene is operably linked to a polynucleotide, the polynucleotide contains a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1 or a functional part or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2 or a functional part or derivative thereof) are proteins expressed in healthy hair cells (e.g., in cochlear and / or vestibular hair cells, such as proteins that play a role in hair cell development, function, regeneration, cell fate determination, survival or maintenance, or proteins that are deficient in subjects with sensorineural hearing loss or vestibular dysfunction) or other therapeutic proteins of interest. Proteins that can be expressed in hair cells using the compositions and methods described herein include 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, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1 and BIP.

[0166] Polynucleotide encoding target protein

[0167] A platform that can be used to achieve therapeutically effective intracellular target protein concentrations in mammalian cells is by stable expression of genes encoding the target protein (e.g., by integration into the nuclear or mitochondrial genome of mammalian cells, or by forming an additional concatemer in the nucleus of mammalian cells). The gene is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. In order to introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. The vector can be introduced into the cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail in, for example, Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0168] The target protein can also be introduced into mammalian cells by targeting the vector containing the gene encoding the target protein to the cell membrane phospholipids. For example, by linking the vector molecule to the VSV-G protein (a viral protein with affinity for all cell membrane phospholipids), the vector can be targeted to the phospholipids on the extracellular surface of the cell membrane. Such constructs can be generated using methods well known to those skilled in the art.

[0169] The recognition and binding of mammalian RNA polymerase to polynucleotides encoding target proteins are important for gene expression. Therefore, sequence elements that show high affinity for transcription factors that recruit RNA polymerase and promote the assembly of transcription complexes at transcription start sites can be included in the polynucleotide. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and final RNA polymerases. Examples of mammalian promoters have been described in Smith et al., Mol. Sys. Biol., 3: 73, published online, and the disclosure thereof is incorporated herein by reference. The promoter used in the methods and compositions described herein is a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1 or a functional part or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2 or a functional part or derivative thereof, optionally comprising a linker located between the first region and the second region.

[0170] Once the polynucleotide encoding the target protein is incorporated into the nuclear DNA of the mammalian cell, the transcription of this polynucleotide can be induced by methods known in the art. For example, the mammalian cell can be exposed to external chemical reagents, such as agents that regulate the combination of transcription factors and / or RNA polymerases with mammalian promoters and thus regulate gene expression to induce expression. Chemical reagents can be used to promote the combination of RNA polymerases and / or transcription factors with mammalian promoters, for example, by removing the repressor protein that has been combined with the promoter. Alternatively, chemical reagents can be used to enhance the affinity of mammalian promoters to RNA polymerases and / or transcription factors, so that in the presence of chemical reagents, the transcription rate of the gene located downstream of the promoter increases. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanism include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA), and can be applied to mammalian cells according to the scheme established to promote gene expression.

[0171] Other DNA sequence elements that may be included in the polynucleotides used in the compositions and methods described herein include enhancer sequences. Enhancers represent another type of regulatory element, which induces conformational changes in the polynucleotides comprising the target gene, so that DNA adopts a three-dimensional orientation that is conducive to the binding of transcription factors and RNA polymerases at the transcription start site. Therefore, the polynucleotides used in the compositions and methods described herein include those encoding the target protein, and additionally include mammalian enhancer sequences. Many enhancer sequences from mammalian genes are now known, and examples include enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers used in the compositions and methods described herein also include those derived from the genetic material of viruses that can infect eukaryotic cells. Examples include SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers on the rear side of the replication origin (bp100-270), and adenovirus enhancers. Other enhancer sequences that induce the activation of eukaryotic gene transcription include CMV enhancers and RSV enhancers. The enhancer can be spliced ​​into the vector containing the polynucleotide encoding the target protein, for example, at the 5' or 3' position of the gene. In a preferred orientation, the enhancer is located at the 5' side of the promoter, which in turn is located 5' relative to the polynucleotide encoding the target protein.

[0172] The nucleic acid vector containing the Myo15 promoter described herein may include a Woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the polyadenylation efficiency of nascent transcripts, thereby increasing the total mRNA amount in the cell. Adding the WPRE to the vector can result in significant improvements in the levels of transgenic expression caused by several different promoters in vitro and in vivo.

[0173] In some embodiments, the nucleic acid vector containing the Myo15 promoter described herein includes a reporter gene sequence, which can be used to verify the expression of the gene operably connected to the Myo15 promoter, for example, in cells and tissues (for example, in hair cells, such as cochlear and / or vestibular hair cells). The reporter gene sequence that can be provided in the transgene includes a DNA sequence encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other substances well known in the art. When associated with a regulatory element (such as the Myo15 promoter) that drives its expression, the reporter gene sequence provides a signal detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic determinations, fluorescence-activated cell sorting determinations and immunoassays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and immunohistochemistry. For example, in the case where the marker sequence is a LacZ gene, the presence of a carrier carrying a signal is detected by a determination for β-galactosidase activity. In the case where the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be visualized by color or light production in a luminometer.

[0174] Methods for delivering exogenous nucleic acids to target cells

[0175] Techniques that can be used to introduce transgenes (such as transgenes operably linked to the Myo15 promoter described herein) into target cells (e.g., mammalian cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells. Mammalian cells (such as human cells) subjected to an external electric field in this way are subsequently susceptible to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. Similar techniques Nucleofection TM The use of applied electric fields to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. TM Protocols that can be used to perform this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0176] Additional techniques that can be used to transfect target cells include extrusion-perforation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. The advantage of this technique is that a vector is not necessary for delivering nucleic acids to cells (such as human target cells). Extrusion-perforation is described in detail, for example, Sharei et al., Journal of Visualized Experiments 81: e50980 (2013), the disclosure of which is incorporated herein by reference.

[0177] Lipofection represents another technique that can be used to transfect target cells. This method includes loading nucleic acids into liposomes, which generally have cationic functional groups, such as quaternary amines or protonated amines, toward the outside of the liposomes. This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately causing the uptake of exogenous nucleic acids, such as by direct fusion of liposomes with cell membranes or by endocytosis of complexes. Lipofection is described in detail in, for example, U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with cell membranes to cause the uptake of exogenous nucleic acids include contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that polynucleotides associate to impart a positive charge that facilitates interaction with cell membranes include activated dendrimers (described, e.g., Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, e.g., Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, e.g., in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0178] Another available tool for inducing uptake of exogenous nucleic acids by target cells is laser transfection, also known as optical transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to and in some cases has been found to be superior to electroporation.

[0179] Puncture transfection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the substrate surface. DNA containing genes intended for intracellular delivery is connected to the surface of the nanostructure. A chip with an array of these needles is then pressed against a cell or tissue. Cells pierced by the nanostructures can express one or more delivered genes. Examples of this technology are described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0180] Magnetorfection can also be used to deliver nucleic acids to target cells. The principle of magnetorfection is to associate nucleic acids with cationic magnetic nanoparticles. Magnetic nanoparticles are made of completely biodegradable iron oxide and coated with specific cationic proprietary molecules that vary according to the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells under the influence of the external magnetic field generated by the magnet. This technology is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0181] Another useful tool for inducing uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves using sound (usually ultrasonic frequencies) to change the permeability of the cell plasma membrane, thereby permeabilizing the cell and allowing the polynucleotide to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0182] Microvesicles represent another potential medium that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles caused by co-overexpression of glycoprotein VSV-G and, for example, genome modification proteins (such as nucleases) can be used to effectively deliver proteins into cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalent incorporation of target polynucleotides (such as genes or regulatory sequences). The use of such vesicles (also referred to as nanovesicles (Gesicles)) in the genetic modification of eukaryotic cells is described in detail, for example, Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract]. In: Methylation changes in early embryonic genes in cancer [Abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; March 13, 2015, Abstract No. 122.

[0183] Vectors for delivering exogenous nucleic acids to target cells

[0184] In addition to achieving high transcription and translation rates, stable expression of exogenous genes in mammalian cells can be achieved by integrating the polynucleotides comprising the genes into the nuclear genome of mammalian cells. A variety of vectors have been developed for delivering and integrating polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are described in, for example, Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). The expression vectors for use in the compositions and methods described herein contain a Myo15 promoter operably linked to a polynucleotide sequence encoding a protein of interest (e.g., a polynucleotide comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1 or a functional part or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2 or a functional part or derivative thereof, the polynucleotide optionally containing a linker located between the first region and the second region), and, for example, additional sequence elements for expressing these agents and / or integrating these polynucleotide sequences into the genome of mammalian cells. Vectors that may contain a Myo15 promoter operably linked to a transgene encoding a target protein include plasmids (e.g., circular DNA molecules that can replicate autonomously inside a cell), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), and viral vectors. Certain vectors that can be used to express a target protein include plasmids containing regulatory sequences that direct gene transcription, such as enhancer regions. Other available vectors for expressing a target protein contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin or nourseoin.

[0185] Viral vectors for nucleic acid delivery

[0186] Viral genomes provide a rich source of vectors that can be used to effectively deliver target genes to the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara, MVA, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, envelope virus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include: avian leukosis-sarcoma, avian C virus, mammalian C, B virus, D virus, oncoretrovirus, HTLV-BLV group, lentivirus, alpha retrovirus, gamma retrovirus, foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, 3rd edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentivirus. Other examples of vectors are described, for example, in US Pat. No. 5,801,030, which is incorporated herein by reference for its disclosure relating to viral vectors for use in gene therapy.

[0187] AAV vectors for nucleic acid delivery

[0188] In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. The rAAV vectors that can be used for the compositions and methods described herein are recombinant nucleic acid constructs comprising: (1) the Myo15 promoter described herein (e.g., a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof, wherein the polynucleotide optionally contains a linker between the first region and the second region), (2) a heterologous sequence to be expressed, and (3) a viral sequence that promotes the stability and expression of the heterologous gene. Viral sequences can include those sequences required for AAV to replicate and package DNA in cis (e.g., functional ITR) into virions. In typical applications, transgenic coding can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate determination, hair cell survival or hair cell maintenance of therapeutic proteins, or in subjects with hereditary hearing loss or vestibular dysfunction forms of wild-type forms of hair cell proteins mutated, which can be used to improve hearing or vestibular function of subjects carrying mutations related to hearing loss, deafness or vestibular dysfunction (e.g., dizziness, vertigo or imbalance). Such rAAV vectors may also contain markers or reporter genes. Available rAAV vectors have one or more AAVWT genes that are completely or partially deleted but still retain functional flanking ITR sequences. AAV ITR may have any serotype suitable for a specific application. For use in the methods and compositions described herein, ITR may be AAV2 ITR. Methods for using rAAV vectors are described in, for example, Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), each of which is incorporated herein by reference for its disclosure relating to AAV vectors for gene delivery.

[0189] The polynucleotides and vectors described herein (e.g., a Myo15 promoter operably linked to a transgenic protein encoding a target protein) can be incorporated into rAAV virions to facilitate the introduction of the polynucleotides or vectors into cells. The capsid protein of AAV constitutes the external non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3 required for virion assembly. The construction of rAAV virions has been described in, for example, US 5,173,414; US 5,139,941; US ​​5,863,541; US ​​5,869,305; US 6,057,152; and US 6,376,237; and Rabinowitz et al., J. Virol. 76: 791 (2002) and Bowles et al., J. Virol. 77: 423 (2003), each of which is incorporated herein by reference for disclosures relating to AAV vectors for gene delivery.

[0190] The rAAV virions that can be used in combination with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb and PHP.S. For targeting hair cells, AAV1, AAV2, AAV2quad (YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8 and PHP.B can be particularly useful. Serotypes evolved for transducing the retina can also be used in the methods and compositions described herein. The construction and use of AAV vectors and AAV proteins of different serotypes are described in, for example, Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), each of which is incorporated herein by reference for its disclosure relating to AAV vectors for gene delivery.

[0191] Also used in combination with the compositions and methods described herein is a pseudotyped rAAV vector. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are derived from capsid genes of serotypes other than a given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.) pseudotyped. The construction and use of pseudotyped rAAV virions are known in the art and described in, for example, Duan et al., J. Virol. 75: 7662 (2001); Halbert et al., J. Virol. 74: 1524 (2000); Zolotukhin et al., Methods, 28: 158 (2002); and Auricchio et al., Hum. Molec. Genet. 10: 3075 (2001).

[0192] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for promoting AAV targeting to specific cell types. The construction and characterization of AAV capsid mutants (including insertion mutants, alanine screening mutants, and epitope tag mutants) are described in Wu et al., J. Virol. 74: 8635 (2000). Other rAAV virions that can be used in the methods described herein include those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25: 436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19: 423 (2001).

[0193] Pharmaceutical composition

[0194] The polynucleotides described herein (e.g., a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, optionally containing a linker located between the first region and the second region) can be operably linked to a transgene (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration to a patient, such as a human patient suffering from sensorineural hearing loss and / or vestibular dysfunction. Pharmaceutical compositions comprising a vector (such as a viral vector) containing a polynucleotide described herein operably linked to a therapeutic transgene 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, such as in the form of a lyophilized formulation or an aqueous solution.

[0195] Mixtures of nucleic acid vectors (e.g., viral vectors) containing polynucleotides described herein (e.g., containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof, the polynucleotide optionally containing a linker between the first region and the second region) operably linked to a therapeutic transgene can be prepared in water suitably mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may contain preservatives to prevent microbial growth. The pharmaceutical form suitable for injection includes sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the preparation can be sterile and can be fluid to the extent that there is easy injectability. The preparation can be stable under manufacturing and storage conditions and can be preserved under conditions that prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The action of microorganisms can be prevented by a variety of antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars or sodium chloride will preferably be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0196] For example, if necessary, the solution containing the pharmaceutical composition described herein can be appropriately buffered, and the liquid diluent can be first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art according to the present disclosure. For example, a dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous infusion fluid or injected at the infusion site of suggestion. According to the disease of the treated subject, a certain dosage variation will necessarily occur. In order to be applied topically to the inner ear, the composition can be formulated to contain synthetic perilymph. Exemplary synthetic perilymph includes 20-200mM NaCl, 1-5mM KCl, 0.1-10mM CaCl2, 1-10mM glucose and 2-50mM HEPE, with a pH between about 6 and 9 and an osmotic degree of about 300mOsm / kg. In any event, the person responsible for administration will determine the appropriate dose for individual subjects. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0197] Treatment

[0198] The compositions described herein can be administered to subjects with sensorineural hearing loss and / or vestibular dysfunction by a variety of routes, such as topical administration to the inner ear (e.g., administration into the perilymph or endolymph, e.g., to the cochlear or vestibular hair cells, e.g., via the oval window, round window, or semicircular canal (e.g., horizontal semicircular canal), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. In any given case, the most appropriate route of administration will depend on the specific composition being administered, the patient, the method of drug formulation, 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 composition can be administered once or more than once (e.g., once a year, twice a year, three times a year, once every two months, or once a month).

[0199] A subject that can be treated as described herein is a subject having or at risk of sensorineural hearing loss and / or vestibular dysfunction (eg, a subject having or at risk of hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects with or at risk of cochlear hair cell damage (e.g., damage associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects with or at risk of vestibular hair cell damage (e.g., damage associated with disease or infection, head trauma, ototoxic drugs, or aging), subjects with or at risk of sensorineural hearing loss, deafness, or auditory neuropathy, subjects with or at risk of vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), subjects with or at risk of tinnitus (e.g., tinnitus alone, or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects with genetic mutations associated with hearing loss and / or vestibular dysfunction, or subjects with a family history of hereditary hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject has hearing loss and / or vestibular dysfunction associated with or caused by loss of hair cells (e.g., cochlear or vestibular hair cells). The methods described herein may include, before treatment or administration with the compositions described herein, a step of screening a subject for a mutation in a gene known to be associated with hearing loss or vestibular dysfunction. Subjects may be screened for gene mutations using standard methods known to those skilled in the art (e.g., gene testing). The methods described herein may also include a step of assessing hearing and / or vestibular function of a subject before treatment or administration with the compositions described herein. Hearing may be assessed using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochlear osmography (ECOG), and otoacoustic emissions. Vestibular function may be assessed using standard tests, such as eye movement tests (e.g., electronystagmography (ENG) or video nystagmography (VNG)), posturography, swivel chair tests, ECOG, vestibular evoked myogenic potentials (VEMPs), and specialist clinical balance tests, such as Mancini and Horak, Eur J Phys Rehabil Med, 46: Those described in 239 (2010). The compositions and methods described herein can also be administered as a prophylactic treatment to patients at risk for developing hearing loss and / or vestibular dysfunction, e.g., patients with a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), patients who carry a genetic mutation associated with hearing loss or vestibular dysfunction but do not yet exhibit hearing loss or vestibular dysfunction, or patients 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).

[0200] The compositions and methods described herein can be used to promote or induce hair cell regeneration (e.g., cochlear and / or vestibular hair cell regeneration) in a subject. Subjects who may benefit from compositions that promote or induce hair cell regeneration include subjects with hearing loss or vestibular dysfunction due to 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 subjects with hair cell abnormalities (e.g., hair cells that do not function normally compared to normal hair cells), hair cell damage (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or a reduction in the number of hair cells caused by genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., increase the survival of damaged hair cells, promote the 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, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs).

[0201] The compositions and methods described herein can also be used to prevent or reduce hair cell damage or death (e.g., cochlear and / or vestibular hair cell damage or death) caused by ototoxic drugs in subjects who have received ototoxic drug treatment or are receiving or are about to receive ototoxic drug treatment. Ototoxic drugs are toxic to the cells of the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness or imbalance), tinnitus, or a combination of these symptoms. Drugs that have been found to have ototoxicity include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin and amikacin), puromycin, antineoplastic drugs (e.g., platinum-containing chemotherapeutic agents, such as cisplatin, carboplatin and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses) and quinine. 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.

[0202] A transgene operably linked to a Myo15 promoter (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 1, or a functional part or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO: 2, or a functional part or derivative thereof) for treating a subject as described herein can be a transgene encoding a protein expressed in healthy hair cells (e.g., in cochlear and / or vestibular hair cells, e.g., a protein that plays a role in hair cell development, function, cell fate determination, regeneration, survival or maintenance, or a protein that is deficient in subjects with sensorineural hearing loss and / or vestibular dysfunction) or another 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 mutation in a particular gene, the transgene can be a wild-type form 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 can be expressed by a transgene operably linked to the Myo15 promoter to treat a subject as described herein include 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, CA CNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRT M2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1 and BIP.

[0203] Treatment may include administering a composition comprising a nucleic acid vector (e.g., an AAV viral vector) containing a Myo15 promoter as described herein in various unit doses. Each unit dose will generally contain a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route of administration and formulation are within the skill of a technician in the clinical field. The unit dose does not need to be administered as a single injection, but may include continuous infusion within a set time period. An injection pump may be used for administration to control the infusion rate so as to minimize damage to the inner ear (e.g., cochlea). In the case where the nucleic acid vector is an AAV vector (e.g., 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), the viral vector may be, for example, about 1 x 10 10 Vector genomes (VG) to 1x 10 15 VG (e.g., 1x 10 10 VG, 2x 10 10 VG, 3x 10 10 VG, 4x10 10 VG, 5x 10 10 VG, 6x 10 10 VG, 7x 10 10 VG, 8x 10 10 VG, 9x 10 10 VG, 1x 10 11 VG, 2x 10 11 VG, 3x10 11 VG, 4x 10 11 VG, 5x10 11 VG, 6x 10 11 VG, 7x 10 11 VG, 8x 10 11 VG, 9x 10 11 VG, 1x 10 12 VG, 2x10 12 VG, 3x 10 12 VG, 4x 10 12 VG, 5x 10 12 VG, 6x 10 12 VG, 7x 10 12 VG, 8x 10 12 VG, 9x 10 12 VG, 1x10 13 VG, 2x 10 13VG, 3x10 13 VG, 4x 10 13 VG, 5x 10 13 VG, 6x 10 13 VG, 7x 10 13 VG, 8x 10 13 VG, 9x10 13 VG, 1x 10 14 VG, 2x 10 14 VG, 3x 10 14 VG, 4x 10 14 VG, 5x 10 14 VG, 6x 10 14 VG, 7x 10 14 VG, 8x10 14 VG, 9x 10 14 VG, 1x10 15 VG) is administered to a patient 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).

[0204] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), 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., hair cell death caused by ototoxic drugs, age-related hair cell death, or noise (e.g., acoustic trauma)-related 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 can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochlear gamma (ECOG), and otoacoustic emissions) and can improve 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 audiometric results obtained before treatment. Vestibular function can be assessed using standard tests for balance and vertigo (e.g., eye movement tests (e.g., ENG or VNG), posturography, swivel chair tests, ECOG, VEMP, and specialist clinical balance tests) and can improve 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 conversation. The compositions described herein may also be administered in an amount sufficient to slow or prevent the development 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, have a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic medications, head trauma, acoustic trauma, or infection), but do not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects who exhibit mild to moderate hearing loss or vestibular dysfunction). Expression of a therapeutic protein encoded by a transgene operably linked to a Myo15 promoter in a nucleic acid vector administered to a subject can be assessed using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting protein or mRNA, and can 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 number of hair cells, the function of hair cells, or the function of a therapeutic protein encoded by a nucleic acid vector administered to a subject can be assessed indirectly based on an audiometric test or a vestibular function test, and can 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 number of hair cells, the function of hair cells, or the function of a therapeutic protein prior to 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 can 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 longer after administration of the compositions described herein. Depending on the dosage and route of administration used for treatment, the patient can be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after administration of the compositions. Depending on the results of the evaluation, the patient may receive additional treatment.

[0205] Pill Box

[0206] The compositions described herein can be provided in a kit for treating sensorineural hearing loss or vestibular dysfunction. The compositions can include polynucleotides described herein (e.g., a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof, the polynucleotide optionally containing a linker located between the first region and the second region), a nucleic acid vector comprising such a polynucleotide, and a nucleic acid vector comprising a polynucleotide described herein operably linked to a transgene encoding a protein of interest (e.g., a protein that can be expressed in hair cells to treat hearing loss and / or vestibular dysfunction). 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 may also include a package insert that instructs the user of the kit (such as a physician) to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

[0207] Example

[0208] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how to use, prepare, and evaluate the compositions and methods described herein and are intended solely as illustrations of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0209] Example 1: Generation of the murine Myo15 promoter

[0210] First, the UCSC Genome Browser (genome.ucsc.edu) was used to identify evolutionarily conserved regions in the vertebrate Myo15 promoter. The region immediately upstream of the Myo15 translation start site (-1 to -1157, SEQ ID NO: 1) and the upstream region containing the non-coding exon 1 of the Myo15 gene (-6755 to -7209, SEQ ID NO: 2) were synthesized and connected together into a single DNA fragment (SEQ ID NO: 13) by de novo gene synthesis. The total size of the truncated Myo15 promoter was 1611 bp, while the total size of the entire genomic region was more than 7000 bp.

[0211] Experiments evaluating the tropism (cell type targeting) and extent and duration of transgene expression achieved by the Myo15 promoter relative to the cytomegalovirus (CMV) promoter in the mouse cochlea yielded results that the Myo15 promoter, but not the CMV promoter, produced selective expression in cochlear hair cells. An AAV construct driving expression of the Aequorea coerulescens green fluorescent protein (AcGFP) using Myo15 was generated to analyze the progression of gene expression relative to a matched standard AAV construct with CMV. Transgene expression was evaluated in experiments in which the virus was delivered to the mouse cochlea of ​​newborn mice, adult mice, and in isolated cochlear explants.

[0212] To evaluate transgene expression, AAV-Myo15-GFP virus was infused into 6-8 week old C57Bl / 6J male mice via the posterior semicircular canal. Mice were allowed to recover from surgery and euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bones were harvested and decalcified in 8% EDTA for 3 days. The cochlea or vestibular system was excised from the decalcified temporal bones and mounted on slides for imaging. Using a ubiquitous promoter, AAV-CMV-GFP induces GFP expression in many cell types within the cochlea, including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia ( Figure 1AUsing a hair cell-specific promoter, AAV-Myo15-GFP was induced to express only in inner and outer hair cells ( Figure 1B In the vestibular system, AAV-Myo15-GFP was induced to express only in vestibular hair cells ( Figure 2 ).

[0213] Example 2: Generation of a minimal Myo15 promoter

[0214] A series of promoters were generated and placed upstream of a fluorescent reporter gene (e.g., GFP, AcGFP, or luciferase). The promoters generated included:

[0215] 1) SEQ ID NO:3 fused to SEQ ID NO:2;

[0216] 2) SEQ ID NO:4 fused to SEQ ID NO:2;

[0217] 3) a fusion of SEQ ID NO:3 and SEQ ID NO:4 fused to SEQ ID NO:2 (e.g., SEQ ID NO:5, 6, or 7);

[0218] 4) SEQ ID NO:8 fused to SEQ ID NO:9 (e.g., SEQ ID NO:10, 11, or 12);

[0219] 5) SEQ ID NO: 1 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);

[0220] 6) SEQ ID NO:3 fused to a fusion of SEQ ID NO:8 and SEQ ID NO:9 (e.g., SEQ ID NO:10, 11, or 12);

[0221] 7) SEQ ID NO: 4 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);

[0222] 8) a fusion of SEQ ID NO:3 and SEQ ID NO:4 (e.g., SEQ ID NO:5, 6, or 7) fused to a fusion of SEQ ID NO:8 and SEQ ID NO:9 (e.g., SEQ ID NO:10, 11, or 12);

[0223] 9) a fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6 or 7);

[0224] 11) SEQ ID NO: 1;

[0225] 12) SEQ ID NO: 2;

[0226] 13) SEQ ID NO: 17;

[0227] 14) SEQ ID NO: 18;

[0228] 15) SEQ ID NO: 17 fused to SEQ ID NO: 18;

[0229] 16) SEQ ID NO: 18 fused to SEQ ID NO: 17;

[0230] 17) SEQ ID NO: 19 fused to SEQ ID NO: 18;

[0231] 18) SEQ ID NO: 20 (e.g., SEQ ID NO: 22, 23, or 24) fused to SEQ ID NO: 21;

[0232] 19) SEQ ID NO: 17 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24);

[0233] 20) SEQ ID NO: 19 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24). The promoter construct is packaged into an AAV serotype capable of transducing hair cells (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, or Anc80L65).

[0234] Viral promoter constructs were used to infect organotypic cochlear explants. After 48 hours of incubation with virus, explants were imaged and analyzed using fluorescence intensity of the reporter gene to assess hair cell-specific expression.

[0235] Example 3: Administration of a composition comprising a nucleic acid vector containing a Myo15 promoter to a subject with sensorineural hearing loss

[0236] According to the methods disclosed herein, a skilled physician in the art can treat a patient, such as a human patient, with sensorineural hearing loss to improve or restore hearing. To this end, a skilled physician in the art can administer to a human patient a composition comprising an AAV vector (e.g., AAV1, AAV2, AAV2quad (YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B) containing a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7, such as SEQ ID NOs 3 and 4) and / or a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, such as SEQ ID NOs 3 and 4). 8 and 9) having a second region with at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), the polynucleotide optionally containing a linker located between the first region and the second region, operably linked to a transgene encoding a therapeutic protein. For example, the polynucleotide operably linked to a transgene encoding a therapeutic protein can be SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 36 or SEQ ID NO: 37. The composition containing the AAV vector can be administered to a patient, for example, by topical administration to the inner ear (e.g., injection into the perilymph) to treat sensorineural hearing loss.

[0237] After the composition is administered to the patient, a skilled practitioner in the art can monitor the expression of the therapeutic protein encoded by the transgene, as well as the improvement of the patient in response to the therapy by a variety of methods. For example, a physician can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochlear osmography (ECOG), and otoacoustic emissions after administering the composition. Compared to the hearing test results before administering the composition, it is found that the patient exhibits an improvement in hearing in one or more tests after administering the composition, which indicates that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0238] Example 4: Murine Myo15 promoter sequence containing portions of SEQ ID NO: 1 and SEQ ID NO: 2 induces transgene expression in hair cells

[0239] Neonatal cochlear explants were established from P0 wild-type CD1 mice. In brief, neonatal mice were euthanized according to the protocol approved by IACUC. The inner ear was removed from the head, and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil, and the top of the cochlear duct was removed to expose the sensory epithelium. Cochlear explants were spread on Mat-tek culture dishes coated with 15% Matrigel, with the luminal side of the cochlear duct facing up. The cochlear explant culture medium composed of DMEM containing 10% FBS and 10ug / mL ciprofloxacin was directly added to the culture. Viruses (AAV 2 / 9-Myo151kb-GFP or AAV2 / 9-Myo15 mini-intron-GFP) containing Myo151kb (SEQ ID NO: 15) promoter or Myo15 mini-intron (SEQ ID NO: 16) promoter were added to the culture medium at a concentration of 1E+10 genome copies / mL. The cultures were incubated in virus-containing medium for 48 h under standard culture conditions at 37°C with 5% CO2. The cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before antibody staining. Antibodies used: rabbit anti-Myo6 (Proteus), donkey anti-rabbit Alexa 568 (Thermo Fisher). Figure 3A-3B As shown, both the Myo15 1 kb (SEQ ID NO: 15) and Myo15 mini-intron (SEQ ID NO: 16) promoters induced the expression of GFP specifically in hair cells.

[0240] Example 5: Myo15 promoter sequence induces transgene expression in hair cells of the mouse cochlea

[0241] Neonatal cochlear explants were established from P0 wild-type B6.CAST-Cdh23Al+ / Kjn mice. Briefly, neonatal mice were euthanized according to an IACUC-approved protocol. The inner ear was removed from the head and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil and the top of the cochlear duct was removed, exposing the sensory epithelium. Cochlear explants were plated on a prepared 3D rat tail collagen matrix with the luminal side of the cochlear duct facing up. Cochlear explant culture medium consisting of DMEM containing 7% FBS and 1U / μL penicillin G was added directly to the culture. Viruses (AAV1-Myo151kb-GFP, AAV1-Myo150.6 kb-GFP, or AAV1-Myo151.2 kb-GFP) containing the murine Myo151kb (SEQ ID NO: 15) promoter, the human Myo150.6 kb (SEQ ID NO: 26), or the human Myo151.2 kb (SEQ ID NO: 25) promoter were added to the culture medium at a concentration of 1E+11 vg / culture. The cultures were incubated in the virus-containing medium for 72 hours under standard culture conditions of 5% CO2 at 37°C. The cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before staining with anti-GFP antibody. The GFP signal as detected with anti-GFP antibody is shown ( Figures 4A-4C ). In separate experiments, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb murine Myo15-GFP was delivered to the cochlea of ​​adult mice via round window injection at a dose of 2.65E+12 vg / ml. Whole mount images ( Figure 4D-4E ) or slice ( Figure 4F-4G ) shows hair cell-specific signals in the apex, middle, and base of the cochlea, as detected with anti-GFP antibody. Figure 4F The arrows in the figure point to GFP-labeled cochlear hair cells. Figure 4F and 4G As shown, expression in the case of the 1.2 kb human Myo15 promoter and the 1 kb mouse Myo15 promoter is primarily restricted to inner hair cells.

[0242] Example 6: Murine Myo15 Promoter Sequence Induces Transgene Expression in the Cochlea of ​​Non-Human Primates in Vivo

[0243] The murine Myo15 promoter specificity was tested in non-human primates. 30 microliters of AAV1-CMV-GFP or AAV1-Myo15 (SEQ ID NO: 13)-GFP were injected into the cochlea through the round window membrane at 15 μl / min. The animals were sacrificed 4 weeks after AAV injection, and the cochlea was harvested and processed as a surface preparation to examine transgene expression without antibody enhancement. AAV1 has high infectivity. Under the ubiquitous CMV promoter, GFP was expressed in hair cells, supporting cells, and fiber cells in the lateral wall of the rhesus monkey cochlea ( Figure 5A In contrast, the 1.6 kb murine Myo15 promoter (SEQ ID NO: 13) restricted GFP transgene expression to hair cells in the cochlea of ​​cynomolgus monkeys ( Figure 5B-5C ).

[0244] Example 7: Murine Myo15 promoter enhances the biological efficacy of AAV-mouse Tmc1 in Tmc1 knockout mice compared to the ubiquitous promoter.

[0245] Tmc1 knockout (KO) mice were anesthetized with isoflurane, shaved, and povidone iodine was applied to the skin. An incision was made below the left ear from above the buccinator muscle to behind the ear. The skin was separated, the muscle was removed and cleaned to expose the posterior semicircular canal. A small hole was made in the semicircular canal using a drill, and the bone was kept dry with a fine cotton swab. A polyamide / polyethylene tube was inserted into the hole and sealed with bone glue. A 1 μl carrier (AAV-CMV1-mouse TMC1 or AAV-Myo15 (SEQ ID NO: 13)-mouse TMC1) with 0.05 μl of trypan blue was delivered to the IL space at a rate of 100 nl / min using a micropump with a Hamilton syringe. After 1 μl was delivered, 5 minutes were passed so that the fluid reached the top of the cochlea and backflow and leakage were prevented. The tube was bent and cut off near the bone. The muscle was pulled back to its original position and the skin was glued together. Mice were given 0.01 cc of meloxicam and then allowed to recover under a heating lamp. Animals were checked for signs of pain or infection for 5 days post-surgery. From 21 to 28 days post-surgery, animals were anesthetized with ketamine and xylazine, and auditory brainstem responses (ABRs) were measured. Figure 6 As shown, the recovery of ABR thresholds was greatly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 compared with AAV-CMV-mouse TMC1.

[0246] Example 8: Human Myo15 promoter sequence induces transgene expression in vivo in the cochlea of ​​non-human primates

[0247] Compositions containing AAV1 vectors carrying the EGFP transgene under the control of the 1.2 kb human myosin 15 promoter (SEQ ID NO: 25) or the 0.6 kb human myosin 15 promoter (SEQ ID NO: 26) were delivered bilaterally into the inner ear of cynomolgus monkeys by round window membrane injection. AAV1-1.2 kb myosin 15-GFP (virus titer 1.06 × 10 13 genome copies / mL) or AAV1-0.6 kb myosin 15-GFP (virus titer was 1.22 × 10 13 Injections (30 μL) of 100 μg / mL of 100 μg / mL were performed at an injection rate of 15 μL / min. Four weeks after injection, the inner ears were removed and the basement membrane surface was prepared. GFP expression was used to monitor myosin 15 promoter activity ( Fig. 7A and 8A , one of the representative ears is shown). AAV1-1.2 kb myosin 15-GFP transduction of the cynomolgus monkey inner ear resulted in GFP expression throughout the basal-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex ( Fig. 7A , frequency in kHz). High-magnification images at 2.8 kHz show that GFP expression was observed in inner hair cells (IHC) and outer hair cells (OHC) ( Figure 7B , upper panel). Similarly, AAV1-0.6 kb myosin 15-GFP transduction of the cynomolgus monkey inner ear resulted in GFP expression throughout the basal-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex ( Fig. 8A , frequency in kHz). High-magnification images at 2.8 kHz show that GFP expression was observed in IHCs and OHCs ( Figure 8B , upper panel). Immunohistochemistry of myosin 7A (Myo7A) was used to visualize hair cells, and nuclei were stained with DAPI ( Figure 7B and Figure 8B , middle and bottom panels).

[0248] Example 9: Myo15 promoter variants induce transgene expression in mouse cochlear explants

[0249] Neonatal cochlear explant culture and viral transduction

[0250] Neonatal cochlear explants were established from P0 wild-type B6.CAST-Cdh23Ahl+ / Kjn mice. Briefly, neonatal mice were euthanized according to a protocol approved by the Institutional Animal Care and Use Committee. The inner ear was removed from the head and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil and the top of the cochlear duct was removed, exposing the sensory epithelium. Cochlear explants were plated on a prepared 3D rat tail collagen matrix with the luminal side of the cochlear duct facing up. Cochlear explant medium consisting of DMEM containing 7% FBS and 1U / μL penicillin G was added directly to the culture. The promoter containing the murine Myo15 1 kb (SEQ ID NO: 15), human Myo15 0.6 kb (SEQ ID NO: 26), human Myo15 1.2 kb (SEQ ID NO: 25), murine Myo15 0.5 kb promoter (SEQ ID NO: 16), murine Myo15 A promoter (SEQ ID NO: 27), murine Myo15 B promoter (SEQ ID NO: 28), murine Myo15 C promoter (SEQ ID NO: 29), murine Myo15A-B promoter (SEQ ID NO: 30), murine Myo15 AC promoter (SEQ ID NO: 31), murine Myo15 BC promoter (SEQ ID NO: 32), murine Myo15 BAC promoter (SEQ ID NO: 33), murine Myo15 CAB promoter (SEQ ID NO: 34), or murine Myo15 BCA promoter (SEQ ID NO: 35). NO:35; AAV1-Myo151kb-GFP, AAV1-Myo150.6kb-GFP, AAV1-Myo151.2 kb-GFP, AAV1-Myo150.5kb-GFP, AAV1-Myo15 A-GFP, AAV1-Myo15B-GFP) viruses were used at 1×10 11 The concentration of 100 vg / culture was added to the medium. The culture was incubated in the virus-containing medium under standard culture conditions at 37°C and 5% CO2 for 72 hours.

[0251] GFP quantification

[0252] After 72 hours of incubation in virus-containing medium, the cultures were fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before anti-Myo7a and / or anti-GFP antibodies. Samples were imaged using a confocal microscope (Zeiss LSM 810) with uniform settings (laser intensity and gain) between samples. ImageJ was used to quantify the GFP intensity values ​​for each cell.

[0253] mRNA quantification

[0254] After 72 hours of cultivation in a medium containing the virus, the culture was quickly frozen on dry ice and RNA was extracted using a PicoPure RNA Isolation Kit (Arcturus cat#0204). For all cDNA synthesis and RT-PCR reactions, GFP and Myo7a mRNA were quantified by qPCR using a Quantinova probe RT-PCR kit (Qiagen cat#208354). GFP expression levels were normalized for Myo7a expression.

[0255] result

[0256] Quantification of GFP expression from the murine Myo15 promoter with hair cell-specific expression using quantitative PCR (qPCR) showed that expression levels of GFP transgenes from the mMyo15 1.6 kb, 1 kb, and 0.5 kb promoters were robust and comparable to each other in the extent of GFP expression ( Fig. 9 Comparison of GFP expression driven by various murine or human Myo15 promoters showed differences in GFP expression in Myo7a-positive hair cells, with the mMyo151 kb promoter producing the highest GFP expression levels per cell ( Fig.10 ).

[0257] Analysis of whole mount images of neonatal cochlear explant cultures infected with a subset of the Myo15 promoter variants described in Table 2 indicated that the promoter sequence of SEQ ID NO:27 (Myo15A) was neither sufficient nor necessary for general promoter activity or hair cell specificity, as Fig. 11B (SEQ ID NO: 27), 11E (SEQ ID NO: 30), 11F (SEQ ID NO: 31) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 27 does provide a splice donor site, which increases the expression level when paired with a splice acceptor site, such as Fig.11A (SEQ ID NO: 16) and 11H (SEQ ID NO: 33), thereby introducing an intron into the promoter sequence. The promoter sequence of SEQ ID NO: 28 (Myo15 B) alone was found to be a core promoter that confers general promoter activity, such as Fig. 11C (SEQ ID NO: 28), but does not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO: 29 (Myo15 C), as shown in FIG. Fig. 11C(SEQ ID NO: 29) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 29 (Myo15 C) was found to be an enhancer that restricts the activity of the core promoter (SEQ ID NO: 28) to inner hair cells and outer hair cells, as shown in FIG. Fig.11G (SEQ ID NO: 32), but is not sufficient for general promoter activity in the absence of the core promoter, such as Fig.11D (SEQ ID NO: 29). The promoter sequences of SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 were reordered into various combinations and produced to result in hair cell-specific promoter activity, such as Fig.11H (SEQ ID NO:33) and 11I (SEQ ID NO:34), although not all rearrangements are tolerated, such as Fig.11J These data are summarized in Table 3 below.

[0258] Table 3. Promoter activity and hair cell specificity of the murine Myo15 promoter

[0259]

[0260] Myo15 promoter activity was further evaluated to determine the expression strength of Myo15 promoter variants that exhibit hair cell specificity, including Myo150.5 kb (SEQ ID NO: 16), Myo15 BC (SEQ ID NO: 32), and Myo15 BAC (SEQ ID NO: 33). The expression levels of the GFP reporter gene from the Myo150.5 kb, Myo15 BC, and Myo15 BAC promoters were robust and comparable to each other. The Myo15 CAB promoter was generated by rearranging the positions of the splice donor and acceptor sites and resulted in cryptic splicing to the middle of the GFP cDNA, as demonstrated by higher expression levels detected with a GFP probe located in the 3' end of the transcript compared to the 5' end of the transcript ( Fig.12 ).

[0261] Quantitative image analysis of GFP expression levels in Myo7a-positive hair cells was performed to assess GFP expression levels per hair cell for various Myo15 promoters, including the 0.5 kb mMyo15 promoter (also known as the Myo15 ABC promoter; SEQ ID NO: 16), the Myo15 AB promoter (SEQ ID NO: 30), the Myo15 AC promoter (SEQ ID NO: 31), the Myo15 BC promoter (SEQ ID NO: 32), the Myo15 A promoter (SEQ ID NO: 27), the Myo15 B promoter (SEQ ID NO: 28), the Myo15 C promoter (SEQ ID NO: 29), the Myo15 BAC promoter (SEQ ID NO: 33), the Myo15C-AB promoter (SEQ ID NO: 34), and the Myo15 BCA promoter (SEQ ID NO: 35). The Myo15 ABC promoter (SEQ ID NO: 16) produced the highest GFP expression per hair cell ( Fig.13A ). Based on tissue autofluorescence (for Fig.13A , <2000 units and for Fig. 13B , <6000 units) to determine the background fluorescence level for each data set.

[0262] Other Implementations

[0263] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be included within the scope of the invention. Other embodiments are in the claims.

Claims

1. A polynucleotide comprising a first region operably linked to a second region, wherein the first region has at least 85% sequence identity with SEQ ID NO: 2 or a functional part or derivative thereof comprising SEQ ID NO: 8 and / or SEQ ID NO: 9, and the second region has at least 85% sequence identity with SEQ ID NO: 1 or a functional part or derivative thereof comprising SEQ ID NO: 3 and / or SEQ ID NO: 4, and the polynucleotide optionally comprises a linker comprising 1 to 100 nucleotides located between the first region and the second region.

2. The polynucleotide of claim 1, wherein the first region comprises or consists of the sequence of SEQ ID NO:

2.

3. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

8.

4. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

9.

5. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:

9.

6. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

10.

7. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

11.

8. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

12.

9. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:

28.

10. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:29.

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