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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• the Atohl protein comprises the sequence of SEQ ID NO: 6 or a variant thereof having one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) conservative amino acid substitutions. In some embodiments, no more than 10% (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or fewer) of the amino acids in the Atohl protein variant are conservative amino acid substitutions. In some embodiments, the Atohl protein has the sequence of SEQ ID NO: 4.
• the nucleic acid vector includes, in 5’ to 3’ order, a first inverted terminal repeat; an SLC6A14 promoter of SEQ ID NO: 1 ; a polynucleotide sequence encoding human Atohl operably linked to the SLC6A14 promoter; a polyadenylation sequence; and a second inverted terminal repeat.
• the nucleic acid vector of the invention includes an SLC6A14 promoter of SEQ ID NO: 1 operably linked to a polynucleotide sequence encoding murine Atohl (e.g., a polynucleotide sequence encoding SEQ ID NO: 6, such as the polynucleotide sequence of SEQ ID NO: 7).
• the Atohl protein has the sequence of SEQ ID NO: 4. In some embodiments, the Atohl protein is encoded by the sequence of SEQ ID NO: 5.
• the invention provides a method of treating a subject having or at risk of developing vestibular dysfunction by administering to an inner ear of the subject an effective amount of the nucleic acid vector or composition of any of the foregoing aspects and embodiments.
• the vestibular dysfunction is vertigo, dizziness, imbalance, bilateral vestibulopathy (also known as bilateral vestibular hypofunction), oscillopsia, or a balance disorder.
• the vestibular dysfunction is age-related vestibular dysfunction, head trauma-related vestibular dysfunction, disease or infection-related vestibular dysfunction, or ototoxic drug-induced vestibular dysfunction.
• the vestibular dysfunction is associated with a genetic mutation.
• the vestibular dysfunction is idiopathic vestibular dysfunction.
• the invention provides a method of inducing or increasing vestibular hair cell regeneration in a subject in need thereof by administering to an inner ear of the subject an effective amount of the nucleic acid vector or composition of any of the foregoing aspects and embodiments.
• the invention provides a method of increasing VSC survival in a subject in need thereof by administering to an inner ear of the subject an effective amount of the nucleic acid vector or composition of any of the foregoing aspects and embodiments.
• the invention provides a method of treating a subject having or at risk of developing oscillopsia by administering to an inner ear of the subject an effective amount of the nucleic acid vector or composition of any of the foregoing aspects and embodiments.
• the nucleic acid vector or composition of the invention is administered into the endolymph. In some embodiments, the nucleic acid vector or composition of the invention is administered to or through the oval window. In some embodiments, the nucleic acid vector or composition of the invention is administered to or through the round window.
• the subject is a human.
• the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of a composition, vector construct, or viral vector described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating vestibular dysfunction, it is an amount of the composition, vector construct, or viral vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, vector construct, or viral vector.
• exon refers to a region within the coding region of a gene, the nucleotide sequence of which determines the amino acid sequence of the corresponding protein.
• exon also refers to the corresponding region of the RNA transcribed from a gene. Exons are transcribed into pre-mRNA and may be included in the mature mRNA depending on the alternative splicing of the gene. Exons that are included in the mature mRNA following processing are translated into protein, wherein the sequence of the exon determines the amino acid composition of the protein.
• heterologous refers to a combination of elements that is not naturally occurring.
• a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.
• nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications.
• this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single-stranded molecule) are provided.
• Polynucleotide sequence as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. , the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.
• the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio.
• transcription regulatory element refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).
• transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, Nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection and the like.
• transduction refers to a method of introducing a vector construct or a part thereof into a cell.
• the vector construct is contained in a viral vector such as for example an AAV vector
• transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or part thereof into the cell genome.
• a transgene such as a transgene operably linked to an SLC6A14 promoter described herein
• a target cell e.g., a mammalian cell
• electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest.
• mammalian cells such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al.
• Magnetofection can also be used to deliver nucleic acids to target cells.
• the magnetofection principle is to associate nucleic acids with cationic magnetic nanoparticles.
• the magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications.
• Their association with the gene vectors (DNA, RNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction.
• the magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.
• sonoporation a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane to permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.
• stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell.
• a variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Examples of expression vectors are described in, e.g., Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006).
• kits for expression of a protein of interest contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
• the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
• viral vectors examples include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
• RNA viruses such as picornavirus and alphavirus
• double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
• herpesvirus e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
• poxvirus e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox
• Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example.
• retroviruses examples include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).
• murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, 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 lentiviruses.
• vectors are described, for example, US Patent No. 5,801 ,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.
• the polynucleotides and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the polynucleotide or vector into a cell (e.g., a VSC).
• the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
• the cap gene encodes three viral coat proteins, VP1 , VP2 and VP3, which are required for virion assembly.
• rAAV virions useful in conjunction 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, 11 , rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eB, and PHP.S.
• AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
• suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
• the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000).
• Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).
• the SLC6A14 promoter is the SLC6A14 promoter of SEQ ID NO: 1 (also represented by nucleotides 219-1977 of SEQ ID NO: 10) and it is operably linked to a polynucleotide sequence encoding human Atohl .
• the polynucleotide sequence encoding human Atohl is SEQ ID NO: 5.
• the polynucleotide sequence that encodes human Atohl is any polynucleotide sequence that, by redundancy of the genetic code, encodes SEQ ID NO: 4.
• the polynucleotide sequence that encodes human Atohl can be partially or fully codon- optimized for expression.
• the vector includes, in 5’ to 3’ order, a first inverted terminal repeat; an SLC6A14 promoter of SEQ ID NO: 1 ; a polynucleotide sequence encoding human Atohl operably linked to the SLC6A14 promoter; a polyadenylation sequence; and a second inverted terminal repeat.
• the polyadenylation sequence has the sequence of nucleotides 3624-3831 of SEQ ID NO: 10.
• the nucleic acid vector includes nucleotides 219-3831 of SEQ ID NO: 10, flanked by inverted terminal repeats.
• the inverted terminal repeats are AAV2 inverted terminal repeats.
• the inverted terminal repeats are any variant of AAV2 inverted terminal repeats that can be encapsidated by a plasmid that carries the AAV2 Rep gene.
• the nucleic acid vector includes nucleotides 219-3831 of SEQ ID NO: 10, flanked by inverted terminal repeats, in which the 5’ inverted terminal repeat has at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
• nucleic acid vector is a viral vector.
• the viral vector is an AAV vector.
• AAV vector has an AAV8 capsid.
• SLC6A14 promoter described herein e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9
• the SLC6A14 promoter is the SLC6A14 promoter of SEQ ID NO: 1 (also represented by nucleotides 219-1977 of SEQ ID NO: 11 ) and it is operably linked to a polynucleotide sequence encoding murine Atohl .
• the polynucleotide sequence encoding murine Atohl is SEQ ID NO: 7.
• the polynucleotide sequence that encodes murine Atohl is any polynucleotide sequence that, by redundancy of the genetic code, encodes SEQ ID NO: 6.
• the nucleic acid vector includes, in 5’ to 3’ order, a first inverted terminal repeat; an SLC6A14 promoter of SEQ ID NO: 1 ; a polynucleotide sequence encoding murine Atohl operably linked to the SLC6A14 promoter; a Woodchuck Posttranscriptional Regulatory Element (WPRE); a polyadenylation sequence; and a second inverted terminal repeat.
• the WPRE has the sequence of SEQ ID NO: 8 or SEQ ID NO: 9.
• the WPRE has the sequence of SEQ ID NO: 8.
• the WPRE has the sequence of nucleotides 3055-3602 of SEQ ID NO: 11.
• the polyadenylation sequence has the sequence of nucleotides 3615- 3822 of SEQ ID NO: 11 .
• the nucleic acid vector includes nucleotides 219-3822 of SEQ ID NO: 11 , flanked by inverted terminal repeats.
• the inverted terminal repeats are AAV2 inverted terminal repeats.
• the inverted terminal repeats are any variant of AAV2 inverted terminal repeats that can be encapsidated by a plasmid that carries the AAV2 Rep gene.
• the nucleic acid vector includes nucleotides 219-3822 of SEQ ID NO: 11 , flanked by inverted terminal repeats, in which the 5’ inverted terminal repeat has at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
• a viral vector of the invention typically requires the use of a plasmid of the invention together with additional plasmids that provide required elements for proper viral packaging and viability (e.g., for AAV, plasmids providing the appropriate AAV rep gene, cap gene and other genes (e.g., E2A and E4)).
• additional plasmids that provide required elements for proper viral packaging and viability (e.g., for AAV, plasmids providing the appropriate AAV rep gene, cap gene and other genes (e.g., E2A and E4)).
• the corresponding sequence in the viral vector can be altered due to the ITRs adopting a “flip” or “flop” orientation during recombination.
• the sequence of the ITR in the transfer plasmid is not necessarily the same sequence that is found in the viral vector prepared therefrom.
• these preparations may contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include 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).
• the formulation may be sterile and may be fluid to the extent that easy syringability exists.
• Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• isotonic agents for example, sugars or sodium chloride.
• 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.
• compositions and methods described herein can be used to induce or increase hair cell regeneration in a subject (e.g., vestibular hair cell regeneration), and/or to induce or increase proliferation of vestibular hair cells and/or VSCs.
• Subjects that may benefit from compositions that promote or induce vestibular hair cell regeneration, vestibular hair cell innervation, and/or vestibular hair cell and/or VSC proliferation include subjects having or at risk of developing vestibular dysfunction as a result of loss of hair cells (e.g., loss of vestibular hair cells related to trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal vestibular hair cells (e.g., vestibular hair cells that do not function properly compared to normal vestibular hair cells), damaged vestibular hair cells (e.g., vestibular hair cell damage related to trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), or reduced vestibular hair cell numbers due to genetic mutations or congen
• Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamycin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, 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.
• aminoglycoside antibiotics e.g., gentamycin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin
• viomycin e.g., antineoplastic drugs (e.g., platinum-containing chemotherapeutic agents, such as cisplatin, carboplatin, and ox
• the methods and compositions described herein can be used to treat bilateral vestibulopathy or oscillopsia due to aminoglycoside ototoxicity (e.g., the methods and compositions described herein can be used to reduce aminoglycoside- induced vestibular hair cell damage or death, or to promote or increase hair cell regeneration and/or hair cell or VSC proliferation in a subject with aminoglycoside-induced bilateral vestibulopathy or oscillopsia).
• the transgene operably linked to an SLC6A14 promoter e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1
• an SLC6A14 promoter e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1
• a transgene that encodes a protein expressed in healthy VSCs e.g., a protein that plays a role in vestibular hair cell development, vestibular hair cell fate specification, vestibular hair cell regeneration, vestib
• the transgene may be selected based on the cause of the subject’s vestibular dysfunction (e.g., if the subject’s vestibular dysfunction is associated with a particular genetic mutation, the transgene can be a wild-type form of the gene that is mutated in the subject, or if the subject has vestibular dysfunction associated with loss of hair cells, the transgene can encode a protein that promotes vestibular hair cell regeneration, vestibular hair cell innervation, or vestibular hair cell and/or VSC proliferation), the severity of the subject’s vestibular dysfunction, the health of the subject’s hair cells, the subject’s age, the subject’s family history of vestibular dysfunction, or other factors.
• the proteins that may be expressed by a transgene operably linked an SLC6A14 promoter for treatment of a subject as described herein include Sox9, Sall2, Camtal , Hey2, Gata2, Hey1 , Lass2,
• Sox10 Gata3, Cux1 , Nr2f1 , Hes1 , Rorb, Jun, Zfp667, Lhx3, Nhlh1 , Mxd4, Zmizl , Myt1 , Stat3, BarhM , Tox, Proxl , Nfia, Thrb, MycM , Kdm5a, Creb314, Etv1 , Peg3, Bach2, IsM , Zbtb38, Lbh, Tub, Hmg20,
• the viral vectors may be administered to the patient at a dose of, for example, from about 1 x 10 9 vector genomes (VG)/mL to about 1 x 10 16 VG/mL (e.g., 1 x 10 9 VG/mL, 2 x 10 9 VG/mL, 3 x 10 9 VG/mL, 4 x 10 9 VG/mL, 5 x 10 9 VG/mL, 6 x 10 9 VG/mL, 7 x 10 9 VG/
• VG/ear 2 x 10 13 VG/ear, 3 x 10 13 VG/ear, 4 x 10 13 VG/ear, 5 x 10 13 VG/ear, 6 x 10 13 VG/ear, 7 x 10 13 VG/ear, 8 x 10 13 VG/ear, 9 x 10 13 VG/ear, 1 x 10 14 VG/ear, 2 x 10 14 VG/ear, 3 x 10 14 VG/ear, 4 x 10 14
• compositions described herein can be provided in a kit for use in treating vestibular dysfunction.
• Compositions may include an SLC6A14 promoter described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1), nucleic acid vectors containing such polynucleotides, and nucleic acid vectors containing a polynucleotide described herein operably linked to a transgene encoding a protein of interest (e.g., a protein that can be expressed in VSCs to treat vestibular dysfunction.
• SLC6A14 promoter described herein e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 8
• the nucleic acid vectors may be packaged in an AAV virus capsid (e.g., AAV1 , AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, 7m8, PHP.B, PHP.eB, or PHP.S).
• AAV virus capsid e.g., AAV1 , AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, 7m8, PHP.B, PHP.eB, or PHP.S.
• the kit can further include a package insert that instructs a 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.
• the percentage of supporting cells with detectable levels of nuclear GFP were comparable between the SLC6A14v2 and SLC6A14v3 promoters (FIG. 2A; points on box plots represent individual utricles). However, the average intensity of nuclear GFP in supporting cells with detectable levels above background was significantly greater for the SLC6A14v3 promoter compared to SLC6A14v2 (FIG. 2B; circles on scatter plots represent individual cells across all samples, black lines are the population average; p ⁇ 0.0001 , Student’s t-test). In addition, the percentage of hair cell nuclei with detectable levels of GFP above background was significantly higher for the SLC6A14v2 promoter compared to SLC6A14v3 (FIG.
• a physician of skill in the art can treat a patient, such as a human patient, with vestibular dysfunction so as to improve or restore vestibular function.
• a physician of skill in the art can administer to the human patient a composition containing an AAV vector (e.g., AAV1 , AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80,
• an AAV vector e.g., AAV1 , AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80,

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• Proteomics, Peptides & Aminoacids (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Display Devices Of Pinball Game Machines (AREA)
• Absorbent Articles And Supports Therefor (AREA)
• Orthopedics, Nursing, And Contraception (AREA)

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• 2022
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