Gene therapy constructs and methods for treating hearing loss

Abstract

Disclosed are compositions and methods that may be useful for treating and / or preventing hearing loss caused by genetic mutations in the STRC gene.The compositions and methods disclosed herein facilitate the delivery of STRC to the inner ear using lentiviral vectors to restore the activity of the STRC gene, promote hair cell survival, prevent further hearing loss, and / or restore hearing in patients suffering from hearing loss, respectively.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 18,8857, filed May 14, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] Technical Field The present disclosure provides compositions and methods useful for treating and / or preventing hearing loss. More specifically, the present disclosure provides compositions and methods useful for treating and / or preventing hearing loss caused by genetic mutations in the STRC gene.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on May 14, 2022, is named BN00002_0051_SL_ST25.txt and is 56KB in size. [Background technology]

[0004] Hearing loss is the most common sensory deficit in humans. According to the 2018 estimates of the degree of disabling hearing loss published by the World Health Organization (WHO), 466 million people worldwide live with disabling hearing loss (432 million adults and 34 million children). The number of people with disabling hearing loss will increase to 630 million by 2030 and to more than 900 million by 2050. More than 90% of people with disabling hearing loss (420 million people) live in low-income areas of the world (WHO global estimates on prevalence of hearing loss, Prevention of Deafness WHO 2018).

[0005] Research has demonstrated that over 50% of prelingual hearing loss is genetic. Such hereditary hearing loss and hearing impairments may be conductive, sensorineural, or a combination of both; syndromic (associated with malformations of the outer ear or other organs or medical problems involving other organ systems) or nonsyndromic (no associated visible abnormalities of the outer ear or any associated medical problems); and prelingual (before language is developed) or postlingual (after language is developed). Furthermore, research has shown that over 70% of hereditary hearing loss is nonsyndromic. Various loci for nonsyndromic hearing loss are referred to as DFN (DeaFNess). The loci are named based on the mode of inheritance: DFNA (autosomal dominant), DFNB (autosomal recessive), and DFNX (X-linked). The numbers following the above names reflect the order of gene mapping and / or discovery. In the general population, the prevalence of hearing loss increases with age. This variation reflects genetic and environmental influences, as well as the interplay between environmental triggers and an individual's genetic predisposition.

[0006] The current treatment options for people with disabling hearing loss are hearing aids or cochlear implants. Cochlear implantation is a common procedure that involves significant medical costs, with lifetime costs exceeding $1,000,000 per patient. The lifetime costs of cochlear implants and hearing aids are prohibitive for most people, especially those living in low-income areas (where the majority of people with disabling hearing loss live). Unfortunately, there are currently no approved therapeutics to prevent or treat hearing loss or hearing loss. Thus, there is an urgent need for treatment options to provide a cost-effective alternative to cochlear implants and hearing aids for hearing loss. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure is based at least in part on the discovery that full-length or near-full-length Stereocilin (STRC) can be incorporated into a lentiviral vector under the control of an inner ear specific promoter (e.g., mouse or human Myo7A promoter) to generate robust expression of STRC in inner ear cells that can rescue phenotypes associated with STRC loss-of-function mutations. The technology herein provides the ability to rescue STRC loss-of-function mutations in mammals (e.g., humans) via gene therapy. The present disclosure provides compositions and methods for restoring STRC function to patients suffering from disorders resulting from STRC mutations.

[0008] In one aspect, the disclosure provides a lentiviral expression vector comprising a nucleic acid sequence encoding Stereocilin (STRC) or a portion thereof; and a promoter operably linked to the nucleic acid sequence.

[0009] In an embodiment, the lentiviral expression vector is a third generation self-inactivating (SIN) lentiviral vector. In an embodiment, a SIN lentiviral vector lacks the long-terminal repeat (LTR) enhancer and promoter elements of wild-type lentivirus.

[0010] In an embodiment, the promoter is selected from the group consisting of STRC promoter, Myo7a promoter, human cytomegalovirus (HCMV) promoter, cytomegalovirus / chicken β-actin (CBA) promoter, and Pou4f3 promoter. In an embodiment, the promoter is Myo7a. In an embodiment, the promoter is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6. Optionally, the Myo7a promoter further comprises a Myo7a enhancer. Optionally, the Myo7a promoter further comprises a Myo7a enhancer. In an embodiment where the promoter is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6, the promoter may optionally further comprise a Myo7a enhancer that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0011] In embodiments, the nucleic acid is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In embodiments, the nucleic acid encodes a polypeptide that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2.

[0012] In one aspect, the disclosure provides a pharmaceutical composition for use in a method for the treatment or prevention of hearing loss comprising a lentiviral expression vector comprising a nucleic acid that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, wherein the nucleic acid sequence is operably linked to a nucleic acid that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4 or SEQ ID NO:6.

[0013] In one aspect, the disclosure provides a lentiviral expression vector comprising the nucleic acid sequence of SEQ ID NO:1 and a cell comprising a promoter operably linked to the nucleic acid.

[0014] In embodiments, the nucleic acid is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1.

[0015] In embodiments, the promoter is selected from the group consisting of the STRC promoter, the Myo7a promoter, the human cytomegalovirus (HCMV) promoter, the cytomegalovirus / chicken beta actin (CBA) promoter or the Pou4f3 promoter.

[0016] In embodiments, the promoter is Myo7a. In embodiments, the promoter is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6.

[0017] In an embodiment, the cell is a stem cell. In an embodiment, the stem cell is an induced pluripotent stem cell.

[0018] In one aspect, the present disclosure provides a method of treating or preventing hearing loss, comprising administering an effective amount of the lentiviral vector of claim 1 to a subject in need thereof.

[0019] In embodiments, the promoter is selected from the group consisting of STRC promoter, Myo7a promoter, human cytomegalovirus (HCMV) promoter, cytomegalovirus / chicken beta actin (CBA) promoter, or Pou4f3 promoter. In embodiments, the promoter is Myo7a. In embodiments, the promoter is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6.

[0020] In embodiments, the expression vector is administered by injection into the inner ear of the subject.

[0021] In embodiments, the injection method is selected from the group consisting of cochleostomy, round window membrane, endolymphatic sac, scala media, canalostomy, scala media via the endolymphatic sac, or any combination thereof.

[0022] In an embodiment, the subject has one or more genetic risk factors associated with hearing loss.

[0023] In embodiments, one of the genetic risk factors is selected from the group consisting of a mutation in the STRC gene.

[0024] In an embodiment, the subject does not exhibit any clinical indicators of hearing loss.

[0025] In one aspect, the disclosure provides a transgenic mouse comprising a deafness-causing mutation / mutation selected from the group consisting of mutations / mutations in the human STRC gene.

[0026] Disclosed herein is an expression vector comprising a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid of SEQ ID NO:1 or SEQ ID NO:2, wherein the nucleic acid sequence is operably linked to a promoter. Also disclosed herein is a pharmaceutical composition for use in a method for treating or preventing hearing loss, comprising an expression vector having a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid of SEQ ID NO:1 or SEQ ID NO:2, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the expression vector is selected from a lentivirus vector, an adeno-associated virus vector, an adenovirus vector, a herpes simplex virus vector, a vaccinia virus vector, or a helper-dependent adenovirus vector. In some embodiments, the vector is a lentiviral or adeno-associated viral vector selected from AAV2, AAV2 / Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80. In some embodiments, the AAV vector may be an AAV50 mixed capsid, which has been shown to provide better transfection of inner and outer hair cells in adult animals when compared to Anc80. In some embodiments, the promoter is selected from any hair cell promoter that drives expression of an operably linked nucleic acid early in development and maintains expression throughout life, such as the STRC promoter, the human cytomegalovirus (HCMV) promoter, the cytomegalovirus / chicken beta actin (CBA) promoter, the Myo7a promoter, or the Pou4f3 promoter.In some embodiments, the enhancer may be a Barhl1 enhancer (see, e.g., Hou et al. (2019) Cell 8(5):458). Examples of endogenous STRC promoters and enhancers are shown in Table 1.

[0027] [Table 1-1] [Table 1-2] [Table 1-3]

[0028] Disclosed herein is a cell having an expression vector comprising a nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO:1, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell.

[0029] Disclosed herein is a method for treating or preventing hearing loss, comprising administering to a subject in need thereof an effective amount of an expression vector comprising a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the expression vector is selected from a lentivirus vector, an adeno-associated virus vector, an adenovirus vector, a herpes simplex virus vector, a vaccinia virus vector, and a helper-dependent adenovirus vector. In some embodiments, the vector is a lentiviral or adeno-associated viral vector selected from AAV2, AAV2 / Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, Anc80, or AAV50. In some embodiments, the promoter is selected from any hair cell promoter that drives expression of an operably linked nucleic acid sequence at early development and maintains expression throughout life, such as the STRC promoter, human cytomegalovirus (HCMV) promoter, cytomegalovirus / chicken beta-actin (CBA) promoter, Myo7a promoter, or Pou4f3 promoter. In some embodiments, the expression vector is administered to the inner ear of the subject, for example, by injection. In some embodiments, the delivery method is selected from the cochlea, the round window membrane, the semicircular canal aperture, or any combination thereof (see, e.g., Erin E. Leary Swan, et al., Inner Ear Drug Delivery for Auditory Applications; Adv Drug Deliv Rev. 2008 December 14; 60(15):1583-1599).In some embodiments, the expression vector is delivered to the scala media via the endolymphatic sac (see, e.g., Colletti V, et al., Evidence of gadolinium distribution from the endolymphatic sac to the endolymphatic compartments of the human inner ear, Audiol Neurootol, 2010;15(6):353-63; Marco Mandala, MD, et al., Induced endolymphatic flow from the endolymphatic sac to the cochlea in Meniere's disease, Otolaryngology-Head and Neck Surgery(2010)143,673-679; Yamasoba T, et al., Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac, Hum Gene Ther. 1999 Mar 20;10(5):769-74). In some embodiments, the subject has one or more genetic risk factors associated with hearing loss. In some embodiments, one of the genetic risk factors is a mutation in the STRC gene. In some embodiments, the mutation in the STRC gene is selected from any one or more STRC mutations known to cause hearing loss (see, for example, Table 4). In some embodiments, the subject does not show any clinical indication of hearing loss.

[0030] In some embodiments, the expression vector described herein is administered as a combination therapy with one or more expression vectors that contain other nucleic acid sequences and / or with one or more other active pharmaceutical agents for treating hearing loss.For example, the combination therapy can include a first expression vector that has the nucleic acid sequence of SEQ ID NO: 1 and a second expression vector that has a nucleic acid sequence, and both expression vectors are administered to a subject as part of the combination therapy for treating hearing loss.

[0031] Disclosed herein is a transgenic mouse having a human STRC gene with a mutation selected from any one or more STRC mutations known to cause hearing loss (see, e.g., Table 4).

[0032] definition By "alteration" is meant an increase or decrease. The alteration can be as small as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or as large as 40%, 50%, 60%, or even as large as 75%, 80%, 90%, or 100%.

[0033] By "biological sample" is meant any tissue, cell, biological fluid, or other material derived from an organism.

[0034] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 70%, more preferably 80% or 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0035] By "fusion protein" is meant an engineered polypeptide that combines sequence elements taken from two or more other proteins.

[0036] As used herein, the terms "transfect," "transfects," "transfecting," and "transfection" refer to the delivery of nucleic acid (usually DNA or RNA) into the cytoplasm or nucleus of a cell, for example, through the use of cationic lipid vehicles and / or by electroporation or other art-recognized transfection means.

[0037] By "transduction" is meant the delivery of a nucleic acid, usually DNA or RNA, into the cytoplasm or nucleus of a cell through the use of viral delivery, e.g., lentiviral delivery vectors / plasmids, or other art-recognized means of transduction.

[0038] The term "plasmid" as used herein refers to an engineered construct consisting of genetic material designed to direct the transformation of a target cell. A plasmid consists of a plasmid backbone. As used herein, a "plasmid backbone" contains multiple genetic elements that are positionally and sequentially oriented with other necessary genetic elements so that the nucleic acid in the nucleic acid cassette can be transcribed and, if necessary, translated in a transfected or transduced cell. As used herein, the term plasmid can refer to a nucleic acid, e.g., DNA, derived from a plasmid vector, cosmid, phagemid, or bacteriophage, into which one or more fragments of nucleic acid encoding a particular gene can be inserted or cloned.

[0039] "Viral vector" as used herein refers to a part of viral genome, such as packaging signal, that is physically integrated into viral particles by the vector, and not simply DNA or a gene located from a part of viral nucleic acid.Thus, although a part of viral genome may be present in the plasmid of the present disclosure, that part does not cause the integration of the plasmid into viral particles, and therefore cannot produce infectious viral particles.

[0040] As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that can replicate and transfer genetic sequences between cells when associated with the appropriate control elements. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0041] As used herein, the term "integrating vector" refers to a vector whose integration or insertion into a nucleic acid (e.g., a chromosome) is achieved via an integrase. Examples of "integrating vectors" include, but are not limited to, retroviral vectors, transposons, and adeno-associated viral vectors.

[0042] As used herein, the term "integrated" refers to a vector that is stably inserted into the genome (ie, chromosome) of a host cell.

[0043] As used herein, the term "exogenous gene" refers to a gene that does not naturally occur in a host organism or cell, or that is artificially introduced into a host organism or cell.

[0044] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a precursor or polypeptide (e.g., STRC). The polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional properties of the full-length or fragment (e.g., improved hair cell survival and hair cell function) are retained. The term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends for a distance of about 1 kb or more on either end, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain the coding region interrupted with non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript, and thus are absent in the messenger RNA (mRNA) transcript. During translation, mRNA functions to specify the sequence or order of amino acids in a nascent polypeptide.

[0045] As used herein, the term "gene expression" refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through gene "transcription" (i.e., through the enzymatic action of RNA polymerase) and, for protein-coding genes, into protein through mRNA "translation." Gene expression can be regulated at many stages of the process. "Upregulation" or "activation" refers to regulation that increases production of gene expression products (i.e., RNA or protein), while "downregulation" or "repression" refers to regulation that decreases production. Molecules (e.g., transcription factors) involved in upregulation or downregulation are often referred to as "activators" and "repressors," respectively.

[0046] When an "amino acid sequence" is recited herein to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms (e.g., "polypeptide" or "protein") are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0047] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, the DNA or RNA sequence codes for an amino acid sequence.

[0048] As used herein, the term "mutant" refers to a protein encoded by a partially homologous nucleic acid, such that the amino acid sequence of the protein is changed.As used herein, the term "mutant" encompasses both the protein encoded by a homologous gene with conservative and non-conservative amino acid substitutions that do not result in a change in protein function, as well as the protein encoded by a homologous gene with amino acid substitutions that cause a decrease in protein function (e.g., null mutation) or an increase in protein function.

[0049] As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The terms also refer to the linking of amino acid sequences in such a manner that a functional protein is produced.

[0050] As used herein, the term "regulatory element" refers to a genetic element that controls some aspects of the expression of nucleic acid sequence.For example, promoter is a regulatory element that facilitates the initiation of transcription of operably linked coding region.Other regulatory elements are splicing signals, polyadenylation signals, termination signals, RNA export elements, internal ribosome entry sites, etc.

[0051] Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription (Maniatis et al., (1987) Science 236:1237). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on which cell type is used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others are functional in a limited subset of cell types (for reviews, see Voss et al., (1986) Trends Biochem. Sci., 11:287; and Maniatis et al., supra). For example, the SV40 early gene enhancer is highly active in a wide variety of cell types from many mammalian species and is widely used for expression of proteins in mammalian cells (Dijkema et al, (1985) EMBO J. 4:761). Two other examples of promoter / enhancer elements active in a wide range of mammalian cell types are those from the human elongation factor 1 alpha gene (Uetsuki et al., (1989) J. Biol. Chem., 264:5791; Kim et al., (1990) Gene 91:217; and Mizushima and Nagata, (1990) Nuc. Acids. Res., 18:5322) and the long terminal repeats of Rous sarcoma virus (Gorman et al., (1982) Proc. Natl. Acad. Sci. USA 79:6777) and human cytomegalovirus (Boshart et al., (1985) Cell 41:521).

[0052] As used herein, the term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions (i.e., functions provided by promoter and enhancer elements, see above for a discussion of these functions). For example, retroviral long terminal repeats contain both promoter and enhancer functions. Enhancers / promoters may be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by genetic engineering (i.e., molecular biology techniques such as cloning and recombination) such that transcription of that gene is directed by the linked enhancer / promoter.

[0053] As used herein, the term "promoter," "promoter element," or "promoter sequence" refers to a DNA sequence that, when linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, but not necessarily, located 5' (i.e., upstream) of the nucleotide sequence of interest that it controls transcription into mRNA and provides a site for specific binding by RNA polymerase and other transcription factors for transcription initiation.

[0054] A promoter can be constitutive or regulatable. When used in relation to a promoter, the term "constitutive" means that the promoter can direct the transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, etc.). In contrast, a "regulatable" promoter is a promoter that can direct a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, etc.), which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus. It is also known in the art that certain promoters confer tissue specificity and / or temporal / developmental specificity to the expression of a nucleic acid sequence under the control of such a promoter.

[0055] As used herein, the term "retrovirus" refers to a retroviral particle that can enter a cell (i.e., the particle contains a membrane-associated protein, such as an envelope protein or viral G glycoprotein, that can bind to the host cell surface and facilitate the entry of the viral particle into the host cell cytoplasm) and integrate the retroviral genome into the host cell's genome (as a double-stranded provirus). The term "retrovirus" encompasses the Oncovirinae (e.g., Moloney murine leukemia virus (MoMLV, also referred to herein simply as "MLV"), Moloney murine sarcoma virus (MoMSV), and mouse mammary tumor virus (MMTV)), Spumavirinae, and Lentivirinae (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus). See, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

[0056] As used herein, the term "retroviral vector" refers to a retrovirus modified to express a gene of interest. Retroviral vectors can be used to efficiently transfer genes into host cells by utilizing the viral infection process. Foreign or heterologous genes cloned (i.e., inserted using molecular biology techniques) into retroviral genome can be efficiently delivered to host cells that are susceptible to infection by retroviruses.

[0057] As used herein, the term "lentiviral vector" refers to a retroviral vector derived from the Lentiviridae family (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus) that can be integrated into non-dividing cells (see, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

[0058] As used herein, the term "adeno-associated virus (AAV) vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, etc. An AAV vector may have one or more of the AAV wild-type genes deleted in whole or in part, preferably the rep gene and / or the cap gene, but retain functional flanking ITR sequences.

[0059] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur within a natural environment.

[0060] As used herein, the term "host cell" refers to any eukaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

[0061] The term "administration" refers to the introduction of a substance into a subject. In general, any route of administration may be utilized, including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into a body compartment. In some embodiments, administration is oral. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous.

[0062] By "agent" is meant any small chemical compound (eg, a small molecule), antibody, nucleic acid molecule, or polypeptide, or fragments thereof, or cell therapy, such as allogeneic transplantation and / or CART cell therapy.

[0063] By "STRC nucleic acid molecule" is meant a polynucleotide that encodes a STRC polypeptide. Exemplary STRC nucleic acid molecules are 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (e.g., NM_153700) (SEQ ID NO:1). JPEG2024518552000005.jpg63150 JPEG2024518552000006.jpg252149 JPEG2024518552000007.jpg116147

[0064] By "STRC polypeptide" is meant a polypeptide or fragment thereof that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (eg, NP_714544.1) (SEQ ID NO:2). JPEG2024518552000008.jpg99147 JPEG2024518552000009.jpg47148

[0065] "STRC genomic sequence" refers to a genomic polynucleotide that encodes a STRC polypeptide. Exemplary STRC genomic sequences are 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (e.g., NC_000015.10) (SEQ ID NO:3): JPEG2024518552000010.jpg171149 JPEG2024518552000011.jpg248146 JPEG2024518552000012.jpg249147 JPEG2024518552000013.jpg250147 JPEG2024518552000014.jpg249147 JPEG2024518552000015.jpg248147 JPEG2024518552000016.jpg48146

[0066] "Human Myo7A promoter" refers to a polynucleotide encoding the human Myo7A promoter region. Exemplary Myo7A promoter nucleic acid molecules are 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (SEQ ID NO:4): JPEG2024518552000017.jpg184148

[0067] By "human Myo7A enhancer" is meant a polynucleotide encoding a Myo7A enhancer region (e.g., the intron 1 enhancer). Exemplary human Myo7A enhancer nucleic acid molecules are 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (SEQ ID NO:5): JPEG2024518552000018.jpg187148

[0068] By "mouse Myo7A promoter" is meant a polynucleotide encoding the mouse Myo7A promoter region. Exemplary Myo7A promoter nucleic acid molecules are 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence (SEQ ID NO:6): JPEG2024518552000019.jpg33148 JPEG2024518552000020.jpg139148

[0069] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to mean within normal tolerances in the art, for example, within 2 standard deviations of the mean. "About" may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0070] In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or apparent from the context (except where such number exceeds 100% of possible values). Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0071] "Control" or "reference" refers to a standard of comparison. Methods for selecting and testing control samples are within the capabilities of one of ordinary skill in the art. Determination of statistical significance is within the capabilities of one of ordinary skill in the art, for example, the number of standard deviations from the mean that constitute a positive result.

[0072] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where explicit disclosure or context clearly dictates otherwise.

[0073] As used herein, the term "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, the subject is a mammal, particularly a primate, especially a human. In some embodiments, the subject is a livestock animal, such as cattle, sheep, goats, cows, pigs, etc.; poultry, such as chickens, ducks, geese, turkeys, etc.; and domesticated animals, especially pets, such as dogs and cats. In some embodiments (e.g., particularly in research situations), the subject mammal is, for example, a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig, such as an inbred pig.

[0074] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive. As used herein, unless otherwise stated or clear from context, the terms "a," "an," and "the" are understood to be singular or plural.

[0075] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and each value is also disclosed herein as "about" that particular value in addition to the value itself. It is also understood that throughout this application, data is provided in several different formats, and this data represents the endpoints and starting points and ranges of any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than, less than, less than, less than, and equal to 10 and 15 are considered to be disclosed, as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0076] Ranges provided herein are understood to be shorthand for all values ​​within the range.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0077] As used herein, the terms "treat," "treating," and "treatment" encompass a variety of activities aimed at a desired change in clinical outcome. For example, the term "treat," as used herein, encompasses any activity aimed at or that achieves a detectable improvement in one or more clinical indicators or symptoms of hearing loss, as described herein.

[0078] The transitional phrase "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to "certain materials or steps" of the claimed embodiment(s) presented in this disclosure and that do not materially affect the basic and novel characteristics.

[0079] The embodiments described and claimed below can be understood in light of the above definitions.

[0080] Other features and advantages of the present disclosure will be apparent from the following description of its preferred embodiments and claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0081] In one aspect, the disclosure provides an expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO:1, and a promoter operably linked to the nucleic acid sequence.

[0082] In some embodiments, the expression vector is a lentiviral vector.

[0083] In some embodiments, the expression vector is an adeno-associated viral vector such as, for example, AAV2, AAV2 / Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, Anc80, or AAV50.

[0084] In some embodiments, the promoter may be the STRC promoter, the Myo7a promoter, the human cytomegalovirus (HCMV) promoter, the cytomegalovirus / chicken beta actin (CBA) promoter, the Barhl1 promoter / enhancer, or the Pou4f3 promoter.

[0085] In one aspect, the disclosure provides a pharmaceutical composition for use in a method for the treatment or prevention of hearing loss, comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO:1, wherein the nucleic acid sequence is operably linked to the nucleic acid.

[0086] In one aspect, the disclosure provides a cell comprising a nucleic acid sequence of SEQ ID NO:1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO:1, and an expression vector comprising a promoter operably linked to the nucleic acid.

[0087] In one aspect, the disclosure provides a method for treating or preventing hearing loss, comprising administering to a subject in need thereof an effective amount of an expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO:1, and a promoter operably linked to the nucleic acid.

[0088] In some embodiments, the expression vector may be a lentiviral vector or an adeno-associated viral vector, such as AAV2, AAV2 / Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, Anc80, or AAV50.

[0089] In some embodiments, the promoter may be a STRC promoter, a Myo6 promoter, a Myo7a promoter, a prestin promoter / enhancer, a Myo15 promoter / enhancer, a human cytomegalovirus (HCMV) promoter, a cytomegalovirus / chicken beta actin (CBA) promoter, a Barhl1 promoter / enhancer, or a Pou4f3 promoter.

[0090] In some embodiments, the cells are stem cells. In some embodiments, the stem cells are induced pluripotent stem cells.

[0091] In some embodiments, the expression vector is administered by injection into the subject's inner ear, hi some embodiments, the injection method is selected from the group consisting of the cochlea foramen, round window membrane, endolymphatic sac, scala centralis, semicircular canal ostium, scala centralis via the endolymphatic sac, or any combination thereof.

[0092] In some embodiments, the subject has one or more genetic risk factors associated with hearing loss.

[0093] In some embodiments, the genetic risk factor may be a mutation in the STRC gene.

[0094] In some embodiments, the subject does not exhibit any clinical indicators of hearing loss.

[0095] In one aspect, the disclosure provides a transgenic mouse comprising a deafness-causing mutation / mutation selected from the group consisting of mutations / mutations in the human STRC gene.

[0096] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of this specification. The above and other objects, features, and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0097] [Figure 1] FIG. 1 shows the location of the Stereocilin (STRC) gene on chromosome 15, 15q13-q21. [Diagram 2] FIG. 2 shows the mRNA transcript map of STRC. [Diagram 3] FIG. 3 shows the mRNA transcript map of the STRC pseudogene. [Figure 4] FIG. 4 shows a linear vector map of an exemplary LV-SIN lentiviral vector, where GOI represents the STRC gene. [Diagram 5] FIG. 5 shows a linear vector map of an exemplary LV-ctrl lentiviral vector. [Figures 6A-6D] Figures 6A-D are a series of dot plots showing dTom expression in HEI-OC1 cells. In particular, the percentage of HEI-OC1 cells expressing the vector-encoded dTomato reporter and STRC protein. Flow cytometry analysis was performed on intracellular staining for dTom expression in non-transduced control (NTC) and cells transduced with LV-ctrl or LV-SIN at MOI 2. The populations shown were pre-gated for live cells using SSC-A / FSC-A signatures, followed by gated for single cells according to FSC-A / FSC-H signatures. Figure 6A shows data for NTC. Figure 6B shows dTom expression at MOI 1.277. Figure 6C shows dTom expression at MOI 3.278. Figure 6D shows dTom expression at MOI 10.279. [Figure 7] Fluorescence images of an exemplary human STRC gene delivery to the inner ear of a mouse via an exemplary embodiment of a gene therapy construct in which a human cytomegalovirus promoter (hcmv-p) / STRC / dTom cassette is incorporated into a third generation lentivirus pseudotyped with the vesicular stomatitis virus (VSV-g) protein. Briefly, STRC transcription is controlled by hcmv-p, and the expressed STRC protein can be easily detected by the dTom tag. Robust delivery to inner hair cells (arrows) and outer hair cells (asterisks) was detected. [Figure 8] Distribution of pseudotyped LV-hcmv-dTom within the inner ear of adult mice. Delivery of 1x106 PU to the posterior semicircular canal of P30 C57Bl / 6 mice. Expression of dTom is seen in all hair cells as well as the spiral ganglion, demonstrating the ability of this vector to target cells targeted by the mutation in STRC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The present disclosure is based, at least in part, on the discovery that full-length or near-full-length Stereocilin (STRC) genes can be incorporated into lentiviral vectors under the control of an inner ear specific promoter (e.g., mouse or human Myo7A promoter) to generate robust expression of STRC in inner ear cells. The technology herein provides the ability to rescue STRC loss-of-function mutations in mammals (e.g., humans) via gene therapy. The present disclosure provides compositions and methods for restoring STRC function to patients suffering from disorders resulting from STRC mutations.

[0099] overview Hearing loss is the most common sensory deficit in humans. According to 2018 estimates of the degree of disabling hearing loss published by the World Health Organization (WHO), 466 million people worldwide live with disabling hearing loss (432 million adults and 34 million children). The number of people with disabling hearing loss will increase to 630 million by 2030 and to more than 900 million by 2050. More than 90% of people with disabling hearing loss (420,000,000 people) live in low-income areas of the world (WHO global estimates on prevalence of hearing loss, Prevention of Deafness WHO 2018).

[0100] Over 50% of prelingual hearing loss is genetic (Centers for Disease Control and Prevention-Heartbeat). Hereditary hearing loss and hearing impairment can be conductive, sensorineural, or a combination of both; syndromic (associated with malformations of the outer ear or other organs or medical problems involving other organ systems) or nonsyndromic (no associated visible abnormalities of the outer ear or any associated medical problems); and prelingual (before language develops) or postlingual (after language develops) (Deafness and Hereditary Hearing Loss Overview; GeneReviews; Richard JH Smith,MD,A Eliot Shearer,Michael S Hildebrand,PhD,and Guy Van Camp,PhD).

[0101] Hearing impairment is a heterogeneous disorder affecting approximately 1 in 1000 newborns. Currently, 42 genes and 69 loci (http: / / hereditaryhearingloss.org) have been implicated in nonsyndromic autosomal recessive hearing loss (locus designation DFNB). In European populations, 20-40% of nonsyndromic hearing loss (NSHL) is due to mutations in GJB2 (MIM:121011) and GJB6 (MIM:604418), which together encompass the DFNB1 locus. With a few exceptions, autosomal recessive NSHL has similar manifestations, in which the hearing loss is severe to profound in a prelingual onset early candidate gene approach that assigned STRC (MIM:606440) to chromosome 15q15.3 encompassing the DFNB16 locus. Stereocilia provide the bridges necessary for longitudinal stiffness and outer hair cell structure, and mechanical deflection opens stereociliary transduction sensitive channels for cell depolarization. Reverse transcriptase polymerase chain reaction (RT PCR) from several mouse tissues showed strong, almost exclusive expression in the inner ear, and knockouts were absent in these important structures (Vona, B et al. “DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics.” Clinical genetics vol. 87,1(2015):49-55.doi:10.1111 / cge.12332.).

[0102] In mixed hearing loss populations, a STRC deletion frequency of more than 1% has been calculated, and the incidence of hearing loss due to STRC is estimated to be 1 in 16,000. Accumulating evidence suggests that DFNB16 constitutes a significant proportion of other genetically heterogeneous etiologies, including NSHL. One challenge that impedes the diagnostic implementation of STRC screening is the presence of a non-processing pseudogene with 98.9% genomic and 99.6% coding sequence identity that resides less than 100 kb downstream from STRC in a region encompassing a segmental duplication with four genes, HISPPD2A (MIM:610979), CATSPER2 (MIM:607249), STRC, and CKMT1A (MIM:613415). Apart from CKMT1A, these pseudogenes carry mutations that render them inactive. Homozygous deletion of STRC and CATSPER2 results in Deafness Infertility Syndrome (DIS; MIM:611102), characterized by hearing loss in both men and women, and exclusive male infertility, as CATSPER2 is required for sperm motility. Not only is it difficult to generate accurate sequencing data that is free of pseudogenes, but it is even more difficult to interpret such data without the usual reliable resources for mutation interpretation, as these databases are "contaminated" with pseudogene data (Vona, B et al. (2015)).

[0103] More than 70% of hereditary hearing loss is nonsyndromic. The various loci for nonsyndromic hearing loss are referred to as DFN (DeaFNess). The loci are named based on the mode of inheritance: DFNA (autosomal dominant), DFNB (autosomal recessive) and DFNX (X-linked). The numbers following the above names reflect the order of gene mapping and / or discovery (Deafness and Hereditary Hearing Loss Overview; GeneReviews; Richard JH Smith,MD,A Eliot Shearer,Michael S Hildebrand, PhD,and Guy Van Camp,PhD). In the general population, the prevalence of hearing loss increases with age. This change reflects genetic and environmental influences, as well as interactions between environmental triggers and an individual's genetic predisposition.

[0104] Sensorineural hearing loss (SNHL) is the most common neurodegenerative disease in humans, with no currently approved pharmacological interventions. SNHL can be caused by genetic disorders as well as acquired through insults such as noise trauma and ototoxicity. Genetic diagnostics have demonstrated that there are at least 100 genes that cause nonsyndromic sensorineural hearing loss, and the majority of causative changes in these genes are single nucleotide variants (SNVs) or small insertions / deletions (indels). In recent years, copy number variants (CNVs) have also been found to play an important role in many human diseases, including neurodevelopmental disorders. CNVs, i.e., changes due to deletions, insertions, or duplications of approximately 1 kb or more in genes, are thought to affect gene expression, phenotypic variation, and adaptation due to gene disruption, which may affect disease traits. More recently, CNVs have been recognized as the primary cause of SNHL. Shearer et al. reported that CNVs were identified in 16 of 89 hearing loss-related genes, with the STRC gene being the most common cause of SNHL4 (Yokota, Yoh et al. “Frequency and clinical features of hearing loss caused by STRC deletions.” Scientific reports vol. 9,1 4408.13 Mar. 2019, doi:10.1038 / s41598-019-40586-7).

[0105] The clinical characteristics of hearing loss patients with detected CNVs were identified by a study of 1,025 subjects (age range 0–70 years, mean age 11.8 years). Based on age at onset, classified as congenital 6 years or younger, 7–18 years, adulthood (>18 years), or unknown, most subjects with a causative STRC deletion were diagnosed with SNHL by adolescence. Causative homozygous STRC deletions were found in 14 of 723 cases (1.94%) classified as isolated autosomal recessive or sporadic, and in 3 of 264 cases (1.14%) with autosomal dominant inheritance. STRC duplications (3 copies) were identified in 19 subjects (1.85%). It was unclear whether the 3 STRC copies were pathogenic or had any effect on the phenotype. Additionally, 27 subjects were identified as having an ST9RC heterozygous deletion, defined as a carrier deletion. The frequency of carrier STRC deletions was 2.63% (27 / 1,025) in the hearing loss cohort, which was identical to the frequency in normal-hearing controls (2.63%, 4 / 152) (Yokota, Yoh et al. (2019)).

[0106] The prevalence of CNVs in STRC among study subjects diagnosed with genetic hearing loss accounted for 5% (17 / 395) of all subjects. Furthermore, when classified based on hearing level as mild-to-moderate or severe-to-profound, the prevalence of causative STRC deletions was 12% (17 / 140) in subjects with mild-to-moderate SNHL. As a result, CNVs in STRC were the second most common cause of mild-to-moderate SNHL after SNVs in GJB2. None of the subjects with severe-to-profound or asymmetric SNHL had disease-causing CNVs in STRC (Yokota, Yoh et al. (2019)).

[0107] Recent advances in genetic and gene therapy techniques indicate that rescue of many recessive forms of hearing loss is possible through gene therapy (Akil et al., 2012; Askew et al., 2015). Long-term gene delivery to the inner ear has been achieved using adeno-associated viral vectors (AAV) (Shu, Tao, Wang, et al., 2016). The first human clinical trial to reverse hearing loss using gene therapy (CGF166) began in June 2014 and was completed in December 2019 (https: / / clinicaltrials.gov / ct2 / show / NCT02132130). This trial evaluated the effect of overexpression of atoh1 in cochlear supporting cells to induce hair cell regeneration. An alternative disease target for translational research in this area is recessive genetic hearing loss affecting defined cell populations within the inner ear. Prevalence of mutations within the general population and retention of normal cellular architecture are additional considerations.

[0108] Currently, there are no approved medications to prevent or treat hearing loss or hearing impairment. The current treatment options for people with disabling hearing loss are hearing aids or cochlear implants. Cochlear implantation is a common procedure with significant medical costs, with lifetime costs exceeding $1,000,000 per patient (Mohr PE, et al. (2000). The societal costs of severe to profound hearing loss in the United States; Int J Technol Assess Health Care; 16 (4): 1120-35). The lifetime costs of cochlear implants and hearing aids are prohibitive for most people, especially those living in low-income areas (where the majority of people with disabling hearing loss live). Treatment options are needed to provide a cost-effective alternative to cochlear implants and hearing aids.

[0109] As described herein, by carefully assessing the incidence of common recessive causes of hearing loss and taking into account the size of the gene and recent advances in viral vector technology (i.e., carrying capacity), it is possible to develop gene therapy programs that have an accessible and fairly common patient population. For example, STRC is the leading cause of congenital hearing impairment worldwide, severe enough to require lifelong use of hearing aids and, in severe cases, cochlear implantation.

[0110] STRC The STRC gene is a known deafness-associated gene that causes mild to moderate hearing loss and is part of a large deletion at the DFNB16 locus on chromosome 15q15.3. The STRC gene is part of a tandem duplication on chromosome 15, with the second copy being a pseudogene. The two copies are separated by less than 100 kb in the telomere-to-centromere direction. The pseudogene is interrupted by a stop codon in exon 20 (e.g., nt4057C>T; aaGln1353Stop).

[0111] STRC contains 29 exons spanning approximately 19 kb. It consists of 1,809 amino acids and contains a putative signal peptide and several hydrophobic segments, suggesting plasma membrane localization. The predicted molecular weight of STRC after signal peptide cleavage is 194 kD.

[0112] The exon map of STRC, including chromosome 15 base pair positions (minus strand), is shown in Table 2. [Table 2]

[0113] The mRNA transcripts found to correspond to the STRC gene are shown below in Table 3. In some embodiments, the STRC gene comprises the Q7RTU9 sequence. [Table 3]

[0114] Stereocilin is expressed in the inner ear, nervous system, and CD14+ cells. The incidence of STRC deletions is estimated to be about 1% to about 5% in the hearing-impaired population (Yokota 2019). Mutations in the STRC gene are associated with DFNB16, an autosomal recessive nonsyndromic form of hearing impairment. DFNB16-related hearing loss is the leading cause of congenital hearing impairment. Clinical features of DFNB16-related hearing loss are as follows (OMIM 603720): Autosomal recessive - Mostly congenital presentation Pre-linguistic onset Moderate to severe hearing loss Affects high frequencies (e.g. high frequency gradients) Most likely to be stable in the long term

[0115] The STRC gene encodes stereocilin, a large extracellular structural protein found in the stereocilia of outer hair cells in the inner ear. It is associated with the horizontal top connector and attachment crown of the tectorial membrane, which are important for proper attachment and positioning of the stereocilia tips (OMIM 606440). Outer hair cell (OHC) bundles are composed of stiff microvilli called stereocilia and are involved in the mechanoreception of sound waves.

[0116] In STRC null mice, the tip links of OHC bundles progressively deteriorate and become completely disconnected from each other, and the tips of the tallest stereocilia do not become embedded in the tectorial membrane. STRC is essential for the formation of horizontal tip-connecting factors that maintain the cohesion of mature OHC bundles (Verpy 2011).

[0117] A STRC deletion frequency of >1% has been calculated in mixed hearing loss populations, and the incidence of STRC hearing loss is estimated at 1 in 16,000. Accumulating evidence suggests that DFNB16 constitutes a valid proportion of otherwise genetically heterogeneous etiologies, including nonsyndromic sensorineural hearing loss (NSHL) (Vona, 2015).

[0118] STRC variants / mutations on chromosome 15 known to cause hearing loss are listed in Table 4. [Table 4]

[0119] Table 5 lists 31 patients with STRC mutations, showing the name of the variant, the gene affected, the protein alteration (if any), the resulting pathology, and their clinical significance. The location of the mutation, the patient's accession number and ID are also provided. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0120] US Patent Application Publication No. 2013 / 0095071 (incorporated herein in its entirety by reference) describes a gene therapy method for restoring age-related hearing loss using a mutant tyrosine adeno-associated virus vector to deliver X-linked inhibitor of apoptosis protein (XIAP) to the round window membrane of the inner ear.However, this publication does not contemplate the delivery of a nucleic acid sequence encoding a functional STRC to prevent or delay the onset of, or restore, hearing loss caused by genetic mutations in the STRC gene, as disclosed herein.

[0121] Furthermore, a key pitfall in the current state of the art for developing clinical gene therapy for hearing impairment is the lack of animal models that reflect human hearing loss. Many of the available mouse models for genetic hearing loss with adult onset in humans exhibit congenital hearing loss, complicating delivery studies. Few models develop genetic hearing loss after hearing development. Delivery of vectors in neonatal mice results in transfection patterns that differ from delivery in adult mice (Shu, Tao, Li, et al., 2016). Novel animal models that can be used to evaluate hearing rescue using different vector systems and gene targets are needed.

[0122] Currently, there is no approved therapeutic treatment for preventing or treating hearing loss or hearing impairment, and no useful preclinical animal model for testing such treatment.The present invention describes compositions and methods for viral gene delivery of STRC to the inner ear to restore the activity of mutant STRC gene, promote hair cell survival, and restore hearing in patients suffering from hearing loss or hearing impairment, as well as cell-based and animal-based models for testing such compositions and methods.

[0123] Hearing loss caused by STRC mutations generally exists in two groups: (i) congenital group, where subjects are born with hearing loss, and (ii) progressive group, where subjects do not have measurable hearing loss at birth, but show progressive hearing loss over a period of time.Thus, in some instances, subjects may have a mutation in the STRC gene (e.g., as detected in a genetic diagnostic test), but do not yet show clinical indicators or symptoms of hearing loss, thus providing an opportunity to begin therapeutic intervention during that time.Thus, in some embodiments, the present invention provides a method for therapeutic intervention during the period of gradual deterioration of hearing.The method of the present invention can be initiated before such period.The method of treating hearing loss provided by the present invention includes, but is not limited to, a method for preventing or delaying the onset of hearing loss or the progression of clinical indicators or symptoms of hearing loss.

[0124] As used herein, the term "hearing loss" is used to describe a diminished ability to hear sound and includes hearing impairment and complete inability to hear.

[0125] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of an active agent described herein that is sufficient to achieve or contributes to the achievement of one or more desired clinical outcomes, for example as described in the above description of "treatment". The appropriate "effective" amount in any individual case can be determined using standard techniques known in the art, such as a dose escalation study.

[0126] As used herein, the term "active agent" refers to a molecule intended to be used in the compositions and methods described herein and intended to be biologically active for purposes of treating, for example, hearing loss (e.g., a lenti- or AAV-derived vector described herein).

[0127] As used herein, the term "pharmaceutical composition" refers to a composition containing at least one active agent or a combination of two or more active agents described herein, together with one or more other ingredients suitable for use in pharmaceutical delivery, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners, excipients, and the like.

[0128] The terms "subject" or "patient", as used interchangeably herein, include mammals, including, but not limited to, humans, non-human primates, rodents (such as rats, mice, and guinea pigs), etc. In some embodiments of the invention, the subject is a human.

[0129] The dosage of the active agent of the present invention can be calculated based on the study in humans or other mammals carried out to determine the efficacy and / or effective amount of the active agent.The dosage and frequency or timing of administration can be determined by methods known in the art and can depend on factors such as the pharmaceutical form of the active agent, the route of administration, whether only one active agent is used or multiple active agents are used (e.g., the dosage of the first active agent required can be lower when such agent is used in combination with a second active agent), and the characteristics of the patient, including age, weight, or the presence of any medical condition that affects drug metabolism.

[0130] In one embodiment, a single dose may be administered, in another embodiment, multiple doses may be administered over a period of time, for example, at specific intervals, such as four times a day, twice a day, once a day, weekly, monthly, etc.

[0131] Clinical Features of Hearing Loss. Hereditary hearing loss and hearing impairments can be conductive, sensorineural, or a combination of both; syndromic (associated with malformations of the outer ear or other organs, or associated with medical problems involving other organ systems) or nonsyndromic (no associated visible abnormalities of the outer ear or any associated medical problems); and prelingual (before language develops) or postlingual (after language develops). (Richard JH Smith, MD, et al.;Deafness and Hereditary Hearing Loss Overview; GeneReviews; Initial Posting:February 14,1999;Last Revision: January 9, 2014.)

[0132] Diagnosis / Testing. Genetic forms of hearing loss should be distinguished from acquired (nongenetic) causes of hearing loss. Genetic forms of hearing loss are diagnosed by otological, audiological, and physical examinations, family history, ancillary tests (e.g., CT scan of the temporal bone), and molecular genetic testing. Molecular genetic testing, which is available for many types of syndromic and nonsyndromic hearing impairment, plays an important role in diagnosis and genetic counseling.

[0133] Selected tests used to measure hearing loss: 1. Distortion Product Otoacoustic Emissions (DPOAEs). Distortion product otoacoustic emissions (DPOAEs) are responses that arise when the cochlea is stimulated simultaneously with two pure tone frequencies whose ratio is between 1.1 and 1.3. Recent studies on the generation mechanism of DPOAEs have highlighted the presence of two important components in the DPOAE response: those generated by intermodulation "distortion" and those generated by "reflections".

[0134] Prevalence of DPOAEs is 100% in normal adult ears. Responses from the left and right ears are often correlated (i.e., very similar). In normal subjects, females have higher amplitude DPOAEs. The aging process affects DPOAE responses by lowering DPOAE amplitude and narrowing the DPOAE response spectrum (i.e., responses at higher frequencies gradually decrease). DPOAEs can also be recorded from other animal species used in clinical research, such as lizards, mice, rats, guinea pigs, chinchillas, chickens, dogs and monkeys. (Otoacoustic Emissions website).

[0135] 2. Auditory Brainstem Response (ABR). Auditory brainstem response (ABR) testing provides information about the inner ear (cochlea) and the brain pathways involved in hearing. This test is sometimes called auditory evoked potential (AEP). This test may be used for children and others who have difficulty with traditional behavioral methods of hearing screening. ABR can also measure WAVE 1 amplitude, which is a measure of neuronal activity involving the synchronous firing of many auditory nerve fibers in the spiral ganglion cells (Verhulst, 2016). ABR is also indicated for people with signs, symptoms, or complaints that suggest some type of hearing loss in the brain or brain pathways. The test is used on both humans and animals. ABR is performed by attaching electrodes to the head, similar to the electrodes placed around the heart when performing an ECG, and recording brainwave activity in response to sound. The person being tested rests quietly or sleeps while the test is being performed. No response is required. ABR can also be used as a screening test in newborn hearing screening programs. When used as a screening test, only one intensity or volume level is checked and the baby either passes or fails the screening. (American Speech-Language-Hearing Association website).

[0136] Clinical manifestations of hearing loss. Hearing loss can be described by the following types and manifestations: type Conductive hearing loss results from abnormalities of the ossicles of the outer and / or middle ear. · Sensorineural hearing loss results from malfunction of the inner ear structures (i.e., the cochlea). Mixed hearing loss is a combination of conductive and sensorineural hearing loss. Central auditory dysfunction results from damage or dysfunction at the level of the eighth cranial nerve, the auditory brainstem, or the cerebral cortex.

[0137] Onset Prelingual hearing loss is present before speech develops. All congenital (present at birth) hearing loss is prelingual, but not all prelingual hearing loss is congenital. Postlingual hearing loss occurs after normal speech has developed. (Richard JH Smith,MD,et al.;Deafness and Hereditary Hearing Loss Overview;GeneReviews;Initial Posting:February 14, 1999;Last Revision:January 9,2014.)

[0138] Severity of Hearing Loss. Hearing is measured in decibels (dB). The threshold or 0 dB mark for each frequency refers to the level at which a normal young adult perceives a tone burst 50% of the time. If an individual's threshold is within 15 dB of the normal threshold, their hearing is considered normal. The severity of hearing loss is graded as shown in Table 6. [Table 6]

[0139] Percent hearing impairment. To calculate the percentage of hearing impairment, subtract 25 dB from the average pure tone average at 500 Hz, 1000 Hz, 2000 Hz, and 3000 Hz. Multiply the result by 1.5 to obtain ear-specific levels. Impairment is determined by weighting the better ear five times as much as the worse ear, as shown in Table 7. Because conversational speech is approximately 50-60 dB HL, calculating functional impairment based on average pure tone hearing can be misleading. For example, a 45 dB hearing loss is much more significant functionally than 30% would imply. A different rating scale is appropriate for young children, where even limited hearing loss can have a significant impact on language development [Northern & Downs 2002]. [Table 7]

[0140] Frequency of hearing loss The frequencies of hearing loss are given as follows: Low (less than 500Hz) ·Intermediate (501~2000Hz) High (over 2000Hz)

[0141] Gene Therapy Gene therapy occurs when DNA is introduced into a patient to treat a genetic disease. This new DNA usually contains a functional gene to correct the effects of a disease-causing mutation in an existing gene. Gene transfer, either for experimental or therapeutic purposes, relies on a vector or vector system that shuttles genetic information into the target cell. The vector or vector system is considered the primary determinant of the efficiency, specificity, host response, pharmacology, and lifespan of the gene transfer reaction. In recent years, the most efficient and effective method to achieve gene transfer is through the use of a vector or vector system based on a replication-defective virus (PCT Publication No. WO2015 / 054653; Methods for predicting ancestral viral sequences and uses thereof).

[0142] Sensory cells in the adult mammalian cochlea lack the capacity for self-repair. Therefore, current therapeutic strategies rely on sound amplification (e.g., hearing aids), better transmission of sound (e.g., middle ear prostheses / active implants), or direct neuronal stimulation (e.g., cochlear implants) to compensate for permanent damage to the primary sensory hair cells or spiral ganglion neurons that form the auditory nerve and relay acoustic information to the brain. Although these approaches are transformative, they are not optimal for restoring the complex human hearing functions that are critical for modern life.

[0143] Introduction of therapeutic genes into the cochlea is believed to further improve the current standard of care for conditions ranging from age-related and environmentally induced hearing loss to genetic forms of hearing loss such as STRC. Over 300 genetic loci have been linked to hereditary hearing loss, and over 70 causative genes have been described (see, for example, Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). Therapeutic success in these approaches depends critically on the safe and efficient delivery of exogenous gene constructs to the relevant therapeutic cellular targets in the organ of Corti (OC) of the cochlea.

[0144] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues (e.g., the cochlea). Such methods can be used to administer nucleic acids encoding components of a nucleic acid targeting system to cells in culture or to a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. (See, e.g., Publication No. JP2022 / 000041A; Systems, methods and compositions for targeted nucleic acid editing).

[0145] vector To date, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, retrovirus, helper-dependent adenovirus and lentivirus have all been tested for cochlear gene delivery. Of these, adeno-associated virus (AAV) has proven to be the most promising, but AAV has limited DNA packaging capacity for genes less than 4.7 kb in length. The STRC gene is 5.5 kb in length. Two different vector systems were tested, one based on a lentiviral vector system and the second based on a dual AAV vector system. The lentiviral vector system disclosed herein has minimal risk of insertional mutagenesis and is pseudotyped to target hair cells. The lentiviral vector system disclosed herein has been tested in the ear for safety and shows consistent delivery to more than 95% of hair cells from base to apex.

[0146] Lentiviral Vectors Lentiviruses belong to the genus Retroviridae. They are unique among retroviruses because they can infect mitotic and postmitotic cells. They can deliver significant amounts of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, and in particular include self-inactivating lentiviral vectors.

[0147] The third generation lentiviral vector system introduced the so-called self-inactivating (SIN) vector. Suitable third generation lentiviral vectors are known in the art and can be prepared and used by those skilled in the art, for example, as described in PCT / EP2021 / 084131, filed December 3, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0148] The optimal method to achieve replication incompetence is to establish a split packaging design and self-inactivation (SIN) due to deletion in the U3 region of 3'LTR. The genes vif, vpr, vpu, nef, and optionally tat should be removed. Specifically, the enhancement for lentivirus system includes a constitutively active heterologous promoter at U3 position, a 5'LTR that includes a repeat region (R) and a U5 region, a 5'UTR that includes a primer binding site (PBS), a splice donor site (SD), a packaging signal (Ψ), a Rev response element, and optionally a splice acceptor (SA) site, an internal enhancer / promoter region operably linked to a cargo sequence, an RNA processing element that optionally includes a woodchuck hepatitis virus posttranscriptional regulatory element (PRE), and a 3'LTR with a deletion (SIN) U3 region, a repeat region (R) and a U5 region.

[0149] These modifications pseudotype the lentiviral vector for its ability to carry foreign viral envelope proteins on its surface. These viral surface glycoproteins regulate viral entry into host cells by interacting with specific cellular receptors to induce membrane fusion and allow cargo load (i.e., STRC) to be delivered to the inner ear of the subject. Specific enhancements allow lentiviral vectors to be pseudotyped with viral envelope glycoproteins that can also bind to LDL receptors or LDL-R family members, such as MARAV-G, COCV-G, VSV-G or VSV-G ts, as well as SLC1A5 receptors, Pit1 / 2 receptors and PIRYV-G receptors.

[0150] An exemplary lentiviral vector that can be used in accordance with the technology of the present specification is the first lentiviral sequence disclosed in part or in its entirety in PCT / EP2021 / 084131. The lentiviral vector can also include a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the first lentiviral sequence disclosed in PCT / EP2021 / 084131. It can also consist of the first lentiviral sequence disclosed in its entirety in PCT / EP2021 / 084131. Alternatively, if the lentiviral vector is pseudotyped with wild-type VSG, VSV-G, or a VSG derivative capable of binding to the LDL receptor or LDL-R family members, and if the wild-type VSV-G is a glycoprotein derived from the Indiana VSV serotype, it may have an amino acid sequence with at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the lentiviral sequences disclosed in PCT / EP2021 / 084131. To achieve higher particle stability upon in vivo administration and to avoid potential recognition by the host's complement system, the thermostable and complement-resistant VSV-G glycoprotein (VSV-G ts) may alternatively be used and may bind to the LDL-R or LDL-R family members.

[0151] The lentiviral vector can be pseudotyped with COCV-G glycoprotein, i.e., glycoprotein derived from Cocal virus. COCV-G can bind to LDL receptor. Alternatively, the glycoprotein used to pseudotype the lentiviral vector of the present invention that can bind to LDL receptor is MARAV-G. The lentiviral vector can also be pseudotyped with viral envelope glycoprotein derived from RD114 glycoprotein (GP) that can bind to SLC1A5 receptor. It can also be glycoprotein derived from BaEV GP that can bind to SLC1A5 receptor.

[0152] Lentiviral vectors can also be pseudotyped with viral envelope glycoproteins that can bind to the Pit1 / 2 receptor. Pit1 and Pit2 are sodium-dependent phosphate transporters that play an important role in phosphate transport to ensure normal cell function. Pit1 and Pit2 also serve as receptors for Gibbon Ape Leukemia Virus (GALV) and Amphotropic Murine Leukemia Virus (A-MuLV), respectively. Thus, the viral envelope glycoproteins can be derived from GALV. GALV GP can bind to the Pit1 / 2 receptor. Alternatively, the viral glycoproteins can be derived from A-MuLV / Ampho. Such Ampho GP can bind to the Pit1 / 2 receptor. They can also be pseudotyped with glycoproteins derived from 10A1 MLV that can bind to the Pit1 / 2 receptor.

[0153] The lentiviral vector can also bind to the Pit1 / 2 receptor and can be pseudotyped with glycoprotein derived from 10A1 MLV. Alternatively, the lentiviral vector can be pseudotyped with PIRYV-G. Thus, the glycoprotein can mediate entry into host cells that can be invaded by PIRYV-G.

[0154] At least four different expression plasmids are provided in the process of packaging lentiviral vector.Lentiviral particles can be provided from the vector plasmid encoding the lentiviral vector genome itself as described above, the packaging plasmid encoding Gag and Pol, the plasmid encoding Rev, and the plasmid encoding at least one of the envelope glycoproteins described herein.The vector plasmid, the plasmid encoding Rev, and / or the plasmid encoding Env can be the nucleic acid sequence disclosed in PCT / EP2021 / 084131.

[0155] The technology herein provides third generation lentiviral vectors comprising a nucleotide sequence encoding a stereocillin gene (STRC) operably linked to a promoter capable of driving high levels of STRC expression in ear cells expressing STRC, as disclosed in PCT / EP2021 / 084131. In some embodiments, the nucleotide sequence encoding STRC may be 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1. In some embodiments, the promoter may be a human Myo7a promoter or a mouse Myo7a promoter. In some embodiments, the promoter may be 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6. In some embodiments, the promoter may be 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4. Those skilled in the art will understand that the Myo7a promoter sequence represented by SEQ ID NO:4 or SEQ ID NO:6 may need to be shortened to facilitate the ability of the promoter:STRC recombinant nucleic acid to be incorporated into the packaging restriction of the lentiviral vector disclosed herein. In particular, within the scope of the present disclosure, it is expressly contemplated that various derivatives of either SEQ ID NO:4 or SEQ ID NO:6, including deletions of the 5' end of certain promoter sequences, may be constructed to facilitate the ability of the Myo7a:STRC recombinant nucleic acid to be incorporated into the lentiviral vector disclosed herein in a manner that allows sufficient packaging of the resulting LV-SIN vector into viral particles.

[0156] The Myo7a promoter has been characterized and the core promoter (e.g., SEQ ID NO: 4) is known to be positively regulated by an enhancer located in the first intron of the Myo7a gene (e.g., Street et al. (2011) A DNA Variant within the MYO7A Promoter Regulates YY1 Transcription Factor Binding and Gene Expression Serving as a Potential Dominant DFNA11 Auditory Genetic Modifier, JBC, 286(17): 15278-15286; Boeda et al. (2001) A specific promoter of the sensory cells of the inner ear defined by trans-Genesis, Human Molecular Genetics, 10(15): 1581-1589), with the sequence of the human version being represented by SEQ ID NO: 5. It is specifically contemplated within the scope of this disclosure that some or all portions of the nucleic acid sequence represented by SEQ ID NO: 5 may be used in combination with the disclosed promoter sequences to promote transcriptional activation of STRC. In some embodiments, the enhancer may be 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5. In some embodiments, SEQ ID NO:4 or SEQ ID NO:6 may be combined with part or all of SEQ ID NO:5 to create a promoter / enhancer combination that may then be operably linked to STRC and incorporated into a third generation lentiviral vector disclosed herein. Without being bound by theory, such promoter / enhancer combinations may further increase the transcriptional activity of STRC in vivo, thereby improving the LV-SIN vectors disclosed herein and enhancing the expression of STRC in patients with disorders associated with STRC mutations. - It is believed that the phenotype can be rescued.

[0157] Adeno-associated virus vector Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of various human diseases. Recent advances in the development of clinically desirable AAV capsids, optimizing genome design utilizing revolutionary biotechnology have substantially contributed to the growth of the gene therapy field. Preclinical and clinical successes in AAV-mediated gene replacement, gene editing and gene silencing have helped AAV become the ideal therapeutic vector of first choice, and two AAV-based therapeutics have gained regulatory approval in Europe or the United States (e.g., Wang, D., Tai, PWL & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. (2019) Nat Rev Drug Discov 18, 358-378). Continued research into AAV biology, as well as an increased understanding of associated therapeutic challenges and limitations, will lay the foundation for future clinical success.

[0158] Adeno-associated viral vector (AAV)-mediated cochlear gene therapy has been applied to animal models of hereditary hearing loss to improve hearing function, but some cochlear cell types have low infection rates. This is due, in part, to the large size of AAV, since only small genes, up to 4.6 kb, can be effectively incorporated into the vector without the risk of producing truncated proteins. For cochlear gene therapy to effectively treat hearing loss, viral vectors with higher efficiency are needed.

[0159] AAV-mediated cochlear gene therapy delivered to the inner ear involves a precise and focused strategy. The organ of Corti (OC) contains two classes of sensory hair cells: inner hair cells (IHCs), which convert mechanical information carried by sound into electrical signals transmitted to neuronal structures, and outer hair cells (OHCs), which serve to amplify and modulate the cochlear response, a process necessary for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells of the spiral limbus that are important for the maintenance of the adjacent tectorial membrane, or supporting cells that have a protective function and can be induced to transdifferentiate into hair cells up to the early neonatal stage.

[0160] Injection into the cochlear duct, which is filled with high-potassium endolymphatic fluid, can provide direct access to hair cells. However, changes in this delicate fluid environment can disrupt the endocochlear potential and increase the risk of injection-related toxicity. The perilymph-filled space surrounding the cochlear duct, scala tympani, and scala vestibuli can be accessed from the middle ear through the oval or round window membrane (RWM). As the only non-bony opening to the inner ear, the RWM is relatively easily accessible in many animal models, and administration of viral vectors using this route is well tolerated. Cochlear implant placement in humans routinely relies on surgical electrode insertion through the RWM.

[0161] Partial rescue of hearing in mouse models of genetic hearing loss was the result of previous studies evaluating AAV serotypes in organotypic cochlear explants and in vivo cochlear injections. In these studies, adeno-associated viruses (AAV) containing ancestral AAV capsid proteins have been observed to transduce OHCs with high efficiency. This finding overcomes the low transduction rates that have limited the successful development of cochlear gene therapy using conventional AAV serotypes. AAV containing ancestral AAV capsid proteins may provide a valuable platform for cochlear gene delivery to IHCs and OHCs, as well as an array of other cochlear cell types compromised by genetic hearing and balance disorders. In addition to providing high transduction rates, AAV containing ancestral AAV capsid proteins have been shown to have a similar safety profile in mice and non-human primates upon systemic injection and are antigenically distinct from circulating AAV, offering potential benefits in terms of pre-existing immunity that limits the efficacy of conventional AAV vectors.

[0162] The viruses described herein that contain ancestral AAV capsid proteins can be used to deliver various nucleic acids to inner ear cells. Representative transgenes that can be delivered to and expressed in inner ear cells include, but are not limited to, transgenes that code for neurotrophic factors (e.g., glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), or heat shock protein (HSP)-70), immunomodulatory proteins, or anti-oncogenic transcripts. In addition, representative transgenes that can be delivered to and expressed in inner ear cells also include, but are not limited to, transgenes that code for antibodies or fragments thereof, antisense, silencing, or long non-coding RNA species, or genome editing systems (e.g., genetically engineered zinc finger nucleases, transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)). Additionally, representative transgenes that can be delivered to and expressed in inner ear cells include the STRCs shown herein, but also include ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DF NB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2 , HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1,MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP are optionally included in a third generation lentiviral vector as disclosed herein.

[0163] Induced pluripotent stem cells (iPSC) Induced pluripotent stem cells (IPS or IPSCs) are stem cells created from adult cells such as skin, liver, stomach or other mature cells through the introduction of genes that reprogram the cell and transform it into a cell with all the characteristics of an embryonic stem cell. The term pluripotency refers to the ability of a cell to give rise to multiple cell types, including all three embryonic lineages that form the body's organs, nervous system, skin, muscle and skeleton.

[0164] Autologous induced pluripotent stem cells (iPSCs) theoretically constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation is a tedious process that poses technical and manufacturing challenges and conceptually impedes any acute treatment modality. Allogeneic iPSC-based or embryonic stem cell-based therapies are relatively easy from a manufacturing point of view and allow the generation of a well-screened, standardized, high-quality cell product. However, due to their allogeneic origin, such cell products are subject to rejection. Reduction or elimination of the antigenicity of the cells can produce a universally tolerated cell product. The potential application of stem cell therapy is broad, since pluripotent stem cells can differentiate into any cell type of the three germ layers. Differentiation can be performed ex vivo or in vivo, by transplanting progenitor cells that continue to differentiate and mature in the organ environment of the transplant site. Ex vivo differentiation allows the researcher or clinician to closely monitor the procedure and ensure that an appropriate population of cells is generated before transplantation.

[0165] However, in most cases, undifferentiated pluripotent stem cells are avoided in clinical transplantation therapies due to their tendency to form teratomas. Rather, such therapies tend to use differentiated cells (e.g., stem cell-derived cardiomyocytes transplanted into the myocardium of patients suffering from heart failure). Clinical application of such pluripotent cells or tissues would benefit from "safety features" that control the proliferation and survival of the cells after transplantation.

[0166] Pluripotent stem cells (PSCs) can be used because they proliferate rapidly and differentiate into many possible cell types. The family of PSCs is generated through different techniques and includes several members with unique immunogenic characteristics. Patient compatibility with engineered cells or tissues derived from PSCs determines the risk of immune rejection and the need for immunosuppression.

[0167] To circumvent the rejection problem, various techniques have been developed to generate patient-specific pluripotent stem cells. These include the transfer of somatic cell nuclei into enucleated oocytes (somatic cell nuclear transfer (SCNT) stem cells), the fusion of somatic cells with ESCs (hybrid cells), and the reprogramming of somatic cells using certain transcription factors (induced PSCs or iPSCs). However, SCNT stem cells and iPSCs may have immune incompatibility with the nuclear or cell donor, respectively, despite their chromosomal identity. SCNT stem cells retain mitochondrial DNA (mtDNA) passed from the oocyte. mtDNA-encoded proteins may act as relevant minor antigens and induce rejection. DNA and mtDNA mutations and genetic instability associated with reprogramming and culture-expansion of iPSCs may also create minor antigens associated with immune rejection. This hurdle reduces the chances of successful large-scale genetic engineering of compatible patient-specific tissues using SCNT stem cells or iPSCs.

[0168] CRISPR / Cas9 gene editing The methods described herein also contemplate the use of CRISPR / Cas9 (clustered regularly interspaced short-palindromic repeats and CRISPR-associated proteins) genome editing to rescue hearing by editing STRC gene mutations.

[0169] This technique has been used to successfully rescue hearing in two genetically deaf mouse models (Tmc1 and Pmca2) (Askew, C et al., Tmc gene therapy restores auditory function in deaf mice; Sci Transl Med. 2015 Jul 8;7(295):295ra108). This technique has primarily been used to target dominant deafness, but can be developed to target recessive deafness and restore hearing in STRC knock-in mouse models and ultimately in humans with deafness caused by mutations in the STRC gene. The use of CRISPR / Cas9 gene editing to repair defective gene sequences is further described in PCT Publication No. WO2016 / 069910, PCT Publication No. WO2015 / 048577, and U.S. Patent Application Publication No. 2015 / 0291966, each of which is incorporated herein by reference in its entirety.

[0170] Conventional molecular biology, microbiology, biochemistry and recombinant DNA techniques within the skill of the art can be used according to the present disclosure. Such techniques are fully explained in the literature and are illustrated in the following examples. The present invention is further described in the following examples, which do not limit the scope of the methods and compositions described in the claims. EXAMPLES

[0171] Example 1: Development of a STRC mutant mouse model The development of a mouse model that is as similar as possible to the human condition is important for early clinical development. Knockout STRC mouse models are available from commercial suppliers and can be used in the experiments described in these examples. In addition, mouse models carrying human mutations known to cause hearing loss have also been generated using CRISPR / Cas9 technology. STRC -The mouse model shows that the human mutation causes hearing loss in mice, making the model valuable for evaluation of gene therapy constructs described below.

[0172] This disclosure provides STRC with human mutations for this study. - A mouse model is provided. The STRC knock-in mouse model disclosed herein provides the ability to test hair cell survival and hearing loss by ABR, DPOAE, and histology. Characterization of the mice includes STRC - Mouse human STRC - The full range of phenotypes, including progressive hearing loss, deterioration of stereocilia tip-links, and detachment of stereocilia to the tectorial membrane, are confirmed, demonstrating the generation of a STRC mouse model for human DFNB16.

[0173] Example 2: Production of lentiviral-STRC constructs for gene therapy As shown in Figure 1, the stereocillin (STRC) gene is located at 15q13-q21 on chromosome 15. Figure 2 shows the mRNA transcription map of STRC. Figure 3 shows the mRNA transcription map of the STRC pseudogene.

[0174] A novel third-generation high-capacity lentiviral vector system was used to deliver the large 5,515 bp STRC cDNA and dTomato reporter gene in one vector. Briefly, the human STRC cDNA sequence (STRC), deposited in NCBI (NM_153700), was flanked by 5' Kozak consensus sequence and SgrAI / AgeI and 3' SalI restriction sites by PCR. The STRC sequence was cloned into a state-of-the-art third-generation self-inactivating (SIN) lentiviral vector carrying the Myo7a promoter, resulting in LV-SIN (shown in FIG. 4).

[0175] FIG. 4 shows a schematic diagram of a typical third generation lentiviral vector containing a gene of interest (GOI) and a promoter (PROM), where the GOI is STRC and the promoter is Myo7a (e.g., SEQ ID NO:4 or SEQ ID NO:6).

[0176] A control vector expressing only the dTomato reporter driven by the SFFV promoter was created by inserting the AgeI- and SalI-flanked dTomato sequence into the vector backbone using the unique AgeI and SalI restriction sites to create pRRL.PPT.SF.dTomato.pre(LV-ctrl), shown in Figure 5 .

[0177] To establish a gene therapy option for STRC mutations, a high-capacity third-generation lentiviral vector was equipped with the large 5,515 bp cDNA sequence of the native STRC isoform. The vector had a self-inactivating (SIN) structure lacking enhancer and promoter elements naturally occurring in the long terminal repeat (LTR). This design confers an improved safety profile by reducing the risk of insertional mutagenesis and allows the use of an internal promoter of choice (e.g., prestin, myosin 6, myosin 7, myosin 15 or hcmv promoter) to drive transgene expression. Here, the myo7a promoter was selected to mediate high-level and sustained cell-type-specific expression of the transgene cassette. To facilitate titration of viral vector particle preparations, as well as identification of successfully transduced cells during in vitro and in vivo applications, the STRC cDNA was linked to a dTomato reporter gene via an internal ribosome entry site (IRES) to generate the lentiviral vector LV-SIN; shown in Figure 4. The counterpart expressing only dTomato served as the reference and control (LV-ctrl) and is shown in Figure 5.

[0178] Transient production using a split packaging system successfully generated lentiviral particles despite the challenging size of the STRC cDNA, and LV titers were in a range sufficient for in vitro and in vivo applications.

[0179] Example 3: Lentiviral STRC constructs are expressed in ear cell lines and organ of Corti cultures The ability of LV-SIN to drive STRC expression was first tested in the HEI-OC1 ear cell line. MYO7A and dTomato were successfully expressed upon in vitro transduction of the cochlea-derived cell line HEI-OC1, one of the few mouse auditory cell lines available for research purposes. HEI-OC1 cells are useful for investigating drug-activated apoptotic pathways, autophagy, aging, mechanisms of cytoprotection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, and the like. According to the techniques herein, HEI-OC1 cells can be used to evaluate the expression of gene constructs in auditory cells. Importantly, HEI-OC1 cells endogenously express prestin, a key motor protein of outer hair cells. In this regard, HEI-OC1 cells serve as a useful in vitro hearing model.

[0180] To assess the functionality of the vector and its ability to transduce inner hair cells, LV-SIN was tested for its in vitro performance using an established hair cell-like cell line, HEI-OC1 (Kalinec et al. (2003) A cochlear cell line as an in vitro system for drug ototoxicity screening. Audiol. Neurotol.).

[0181] HEI-OC1 cells were plated at 3 × 10 per well in a 24-well plate the day before transduction. 4Cells were seeded at 1000 x g for 30 min at 4 °C. Triplicate wells were harvested for counting to determine cell number at the time of transduction, and the volume of viral vector supernatant was calculated based on the vector titer to apply a defined multiplicity of infection (MOI), i.e., a defined number of particles per cell seeded. The transduction procedure followed the same protocol as described in the titration above. The percentage of cells expressing the vector-encoded dTomato reporter protein was assessed by flow cytometry as described in the titration above.

[0182] Cells were harvested using trypsin-assisted detachment and pelleted by centrifugation at 400xg for 5 min. The pellet was resuspended in 500 μL of Fixation Buffer (Cat. No. 420801, BioLegend, San Diego, CA, USA) and the cells were incubated at room temperature for 20 min. Samples were pelleted again and washed with 1 mL of FACS buffer, followed by three cycles of resuspension in 1× Intracellular Staining Perm Wash Buffer (Cat. No. 421002, BioLegend) and centrifugation at 400xg for 5 min. Incubation with the primary antibody polyclonal rabbit anti-myosin VIIA (Cat. No. 25-6790, Proteus BioSciences Inc., Ramona, CA, USA) was performed at 1:300 dilution in 1× Intracellular Staining Perm Wash Buffer for 20 min at room temperature, followed by two washes with 1× Intracellular Staining Perm Wash Buffer. Incubation with secondary antibody Alexa Fluor® 488 AffiniPure donkey anti-rabbit IgG (H+L) (cat. no. 711-545-152, Jackson ImmunoResearch Europe Ltd, Ely, UK) was performed at 1:800 dilution in 1× Intracellular Staining Perm Wash Buffer for 20 min at room temperature in the dark. After two washes with 1× Intracellular Staining Perm Wash Buffer, cell pellets were resuspended in FACS buffer, processed on a CytoFLEX S flow cytometer, and analyzed using CytExpert software.

[0183] Upon transduction at different multiplicities of infection (MOI), i.e., application of a defined number of viral vector particles per seeded cell, no significant differences in the percentage of successfully transduced dTomato-positive cells were observed by flow cytometry analysis between LV-SINLV-SIN and LV-ctrl across all MOIs tested. Figure 6A-D are a series of dot plots showing dTom expression in HEI-OC1 cells. In particular, the percentage of HEI-OC1 cells expressing the vector-encoded dTomato reporter and STRC protein. Flow cytometry analysis was performed on intracellular staining for dTom expression in non-transduced control (NTC) and cells transduced with LV-ctrl or LV-SIN at a series of different MOIs. The indicated populations were pre-gated for live cells using SSC-A / FSC-A signatures and subsequently gated for single cells according to FSC-A / FSC-H signatures. Figure 6A shows data for NTC. Figure 6B shows dTom expression at MOI 1.277. Figure 6C shows dTom expression at MOI 3.278. Figure 6D shows dTom expression at MOI 10.279. This confirmed that the transduction efficiency of the lentiviral vector encoding the large STRC cDNA was comparable to the smaller vector.

[0184] Visualization by immunofluorescence microscopy or flow cytometry revealed low levels of endogenous STRC expression in non-transduced HEI-OC1 cells, with no signal for dTomato (Figure 6A-D). In summary, despite the large size of the STRC transgene, we were able to produce fully functional LV vector particles that successfully transduced and expressed STRC in otic target cells.

[0185] Example 4: Lentiviral STRC constructs are expressed in the inner ear of mice After confirming that STRC could be delivered by and expressed from LV-STRC, the ability of this STRC to be properly expressed in vivo was examined. 16-day-old adult C57BL / 6 mice were anesthetized with an intraperitoneal (IP) injection of a mixture of ketamine (150 mg / kg), xylocaine (6 mg / kg) and acepromazine (2 mg / kg) in 0.9% sodium chloride. A dorsal postauricular incision was made to expose the posterior semicircular duct. A microdrill was used to create a duct opening exposing the perilymphatic space. Subsequently, 1 μL of vector was injected using a Hamilton microsyringe with a 0.1 μL graduation and a 36-gauge needle. The duct opening was sealed with bone wax and the animals were allowed to recover.

[0186] LV-SIN was injected into the inner ear of wild-type mice as described above to evaluate the performance of LV-STRC to drive in vivo expression of human STRC. As shown in Figure 7, STRC (visualized by dTom expression) was strongly expressed within the inner ear of mice. Notably, robust expression was observed in inner hair cells (arrows) and detected in outer hair cells (asterisks). The characteristics of successful packaging and efficient in vivo delivery of STRC in the absence of adverse effects on wild-type mice indicate that LV-SIN is a suitable candidate for in vivo gene therapy of STRC-associated genetic disorders.

[0187] FIG. 8 shows the distribution of pseudotyped LV-hcmv-dTom in the inner ear of adult mice. 6 Delivery of PU.dTom expression was seen in all hair cells as well as the spiral ganglion, demonstrating the ability of this vector to target cells targeted by the mutation in STRC.

[0188] Example 5: Study of LV-SIN in Hearing Recovery LV-SIN was used to identify newborn STRC -Inject into the inner ear of mutant mice. Analyses will be performed on LV-GFP / dTom injected and control mice, which may include hearing tests, cellular and molecular studies, as well as long-term effects. LV-SIN may be evaluated at the cellular level to determine if it promotes hair cell survival at 1 month of age. In control mutant ears injected with LV-GFP / dTom, there is expected to be a loss of hair cells at this time point. In contrast, LV-SIN injected hair cells are expected to survive. The injection procedure (cochleostomy, round window membrane, canaliculoplasty) and the dose for better hearing recovery. Importantly, injections may be performed in adult (1-6 months old) mice to assess the potential for hearing recovery. Adult injection results will be compared to neonatal results, which will provide information about the time window in which the intervention is still effective.

[0189] Example 6: Study of patient-derived hair cells Induced pluripotent stem cell (iPS) cells One important aspect of the study is to demonstrate that the techniques disclosed herein can be effective on human hair cells. Since human temporal bones are not available for study, iPS cell lines are established from the patient's iPS cells using fibroblasts from the patient as well as fibroblasts from a control family member. Fibroblasts are taken from the patient with the most frequent mutation and an iPS cell line is established. The iPS cell line is differentiated into inner ear cells, including hair cells. In this culture system, LV-SIN is used to infect iPS-derived hair cells. The infected hair cells are studied for survival and hair cell transduction by patch clamp technique. It is expected that there will be an improvement in hair cell survival and hair cell function compared to uninfected and untreated control hair cells. This study provides an opportunity to evaluate the efficacy of LENTI-STRC infection and expression of the STRC gene in human hair cells. Such an achievement is a demonstration that defective human hair cells can be treated with LV-SIN, which is one major step forward to future clinical trials.

Claims

1. A nucleic acid sequence encoding a stereocillin (STRC), or a portion thereof; and a promoter operably linked to the nucleic acid sequence. Lentiviral expression vectors.

2. The lentiviral expression vector is a third generation self-inactivating (SIN) lentiviral vector, and optionally 2. The lentiviral expression vector of claim 1, wherein the SIN lentiviral vector lacks the long terminal repeat (LTR) enhancer and promoter elements of a wild-type lentivirus.

3. 2. The lentiviral expression vector of claim 1, wherein the promoter is selected from the group consisting of STRC promoter, Myo7a promoter, human cytomegalovirus (HCMV) promoter, cytomegalovirus / chicken beta actin (CBA) promoter and Pou4f3 promoter.

4. The promoter is Myo7a, and optionally further comprises a Myo7a enhancer, and optionally 4. The lentiviral expression vector of claim 3, wherein the promoter is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:6, and optionally further comprises a Myo7a enhancer that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

5.

5. The lentiviral expression vector of claim 1 , wherein the nucleic acid is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

1.

6. The lentiviral expression vector of claim 1 , wherein the nucleic acid encodes a polypeptide that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

2.

7. A pharmaceutical composition comprising an effective amount of the lentiviral expression vector of claim 1, for treating or preventing hearing loss when administered to a subject.

8. A pharmaceutical composition for the treatment or prevention of hearing loss, comprising a lentiviral expression vector comprising a nucleic acid that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, wherein the nucleic acid sequence is operably linked to a nucleic acid that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4 or SEQ ID NO:

6.

9. A cell comprising a lentiviral expression vector comprising the nucleic acid sequence of SEQ ID NO:1 and a promoter operably linked to said nucleic acid, optionally (a) the nucleic acid is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1; and / or (b) the promoter is selected from the group consisting of STRC promoter, Myo7a promoter, human cytomegalovirus (HCMV) promoter, cytomegalovirus / chicken beta actin (CBA) promoter, or Pou4f3 promoter.

10. The promoter is Myo7a, and optionally The cell of claim 9, wherein the promoter of (b) is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or SEQ ID NO:

6.

11. The cell of claim 10, wherein the cell is a stem cell, and optionally The cell of claim 9 , wherein the stem cell is an induced pluripotent stem cell.

12. A pharmaceutical composition comprising an effective amount of the lentiviral expression vector of claim 7, (a) the expression vector is administered by injection into the inner ear of the subject; (b) the injection method is selected from the group consisting of the cochlear foramen, the round window membrane, the endolymphatic sac, the scala centralis, the semicircular canal ostium, the scala centralis via the endolymphatic sac, or any combination thereof; and / or (c) the subject has one or more genetic risk factors associated with hearing loss; and optionally, one of the genetic risk factors is selected from the group consisting of a mutation in the STRC gene; and further optionally, The pharmaceutical composition, wherein the subject does not exhibit any clinical indicators of hearing loss.

13. A transgenic mouse comprising a deafness-causing mutation / mutation selected from the group consisting of mutations / mutations in the human STRC gene.

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