Stereocylin promoter and uses thereof

Abstract

The present disclosure provides a stereocillin (STRC) promoter and vectors containing same that can be used to drive expression of a desired expression product in hair cells that endogenously express STRC, including hair cells of the cochlea and vestibule. The STRC promoter described herein can be operably linked to a polynucleotide, such as a transgene, that encodes a heterologous expression product and used to treat subjects having or at risk of developing hearing loss or vestibular dysfunction.

Description

[Background technology]

[0001] Hearing loss is a major public health problem, estimated to affect nearly 15% of school-age children and one in three people by age 65. The most common type of hearing loss is sensorineural hearing loss, which is caused by abnormalities in the cells of the inner ear, such as the cochlear hair cells, or abnormalities in the neural pathways that project from the inner ear to the brain. Sensorineural hearing loss is often acquired and has a variety of causes, including acoustic trauma, disease or infection, head trauma, ototoxic drugs, and aging. There are also genetic causes of sensorineural hearing loss, such as mutations in genes involved in the development and function of the inner ear. Mutations in over 90 such genes have been identified, including mutations inherited in an autosomal recessive, autosomal dominant, and X-linked manner. One such autosomal recessive type of sensorineural hearing loss is associated with mutations in the STRC gene. Stereocylin is a large protein encoded by the STRC gene on chromosome 15q15, which contains 29 exons spanning approximately 19 kb of the genome. The STRC gene is tandemly duplicated, in which the second copy contains a premature stop codon in exon 20, thereby giving rise to a STRC pseudogene. Previous studies have identified STRC mutations in families with autosomal recessive nonsyndromic sensorineural hearing loss (Non-Patent Document 1). Expression of stereocillin protein is restricted to the stereocilia of hair cell bundles, and stereocillin protein is thought to form the distal lateral connectors and tectorial adhesion crowns required for the normal functioning of the hearing apparatus (Non-Patent Document 2, Non-Patent Document 3). Stereocilin-deficient mice have been shown to exhibit abnormal hair cell bundles with adhesion defects and hearing impairment (Non-Patent Document 4).

[0002] Factors that disrupt the development, survival, or integrity of cochlear hair cells, such as genetic mutations, disease or infection, ototoxic drugs, head trauma, and aging, can affect vestibular hair cells as well, and therefore also contribute to vestibular dysfunction, such as vertigo, dizziness, and imbalance. Indeed, patients with mutations that disrupt hair cell development or function may present with both hearing loss and vestibular dysfunction, or either disorder alone. Approximately 35% of adults in the United States over the age of 40 exhibit balance disorders, and this percentage increases dramatically with age, resulting in disruptions to daily activities, declines in mood and cognitive function, and an increased incidence of falls in the elderly.

[0003] In recent years, efforts to treat hearing loss and vestibular dysfunction have increasingly turned to gene therapy as a possible solution, but few approaches exist to specifically target the hair cells of the cochlea or vestibular system, which are frequently involved in hearing loss and vestibular dysfunction, respectively. New hair cell-targeting therapies are needed to treat sensorineural hearing loss and vestibular dysfunction. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Verpy et al., Nat. Genet. 29:345-9 (2001) [Non-Patent Document 2] Avan et al.,PNAS 116:25948-57(2019) [Non-Patent Document 3] Verpy et al.,J.Comp.Neurol.519:194-210(2011) [Non-Patent Document 4] Verpy et al.,Nature 456:255-8(2008) Summary of the Invention

[0005] The present invention provides compositions and methods for promoting expression of a gene of interest in a particular cell type, for example, a gene that promotes or improves hair cell function, regeneration, or survival. The compositions and methods described herein relate to polynucleotides that can induce expression of a transgene in cochlear and vestibular hair cells of the inner ear. The polynucleotides described herein can be operably linked to a polynucleotide encoding a desired expression product, for example, a protein or an inhibitory RNA, and can be administered to a subject, such as a human subject, to treat or prevent hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular disorders, oscillopia, or balance disorders). The present invention also provides a two-vector system including a first nucleic acid vector containing a polynucleotide described herein operably linked to a polynucleotide encoding the N-terminal portion of a stereocillin protein and a second nucleic acid vector containing a polynucleotide encoding the C-terminal portion of a stereocillin protein, which can be used to treat a subject having or at risk of developing hearing loss or vestibular dysfunction associated with a mutation in the stereocillin gene (STRC).

[0006] In a first aspect, the invention provides a polynucleotide comprising a STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2, operably linked to a polynucleotide encoding a heterologous expression product.

[0007] In another aspect, the invention provides a polynucleotide comprising a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48, or a functional portion thereof including nucleotides 280-560 of SEQ ID NO:48, operably linked to a polynucleotide encoding a heterologous expression product.

[0008] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide according to any of the previous aspects. In another aspect, the invention provides a nucleic acid vector containing a STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0009] In another aspect, the invention provides a nucleic acid vector containing a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48 or a functional portion thereof including nucleotides 280-560 of SEQ ID NO:48.

[0010] In some embodiments of any of the aforementioned aspects, the STRC promoter has at least 85% sequence identity to SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0011] In some embodiments of any of the aforementioned aspects, the STRC promoter consists of SEQ ID NO:1. In some embodiments of any of the foregoing aspects, the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0012] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 252-537 of SEQ ID NO:2. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 120-537 of SEQ ID NO:2. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 35-530 of SEQ ID NO:2.

[0013] In some embodiments of any of the aforementioned aspects, the STRC promoter consists of SEQ ID NO:2. In some embodiments of any of the aforementioned aspects, the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48.

[0014] In some embodiments of any of the aforementioned aspects, the STRC promoter has the sequence of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 280-560 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 280-564 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 124-560 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 124-564 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 1-560 of SEQ ID NO:48.

[0015] In some embodiments of any of the aforementioned aspects, the STRC promoter is operably linked to a polynucleotide encoding a heterologous expression product. In some embodiments of any of the foregoing aspects, the heterologous expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA. In some embodiments, the protein is Actin Gamma 1 (ACTG1), Fascin Actin-Bundling Protein 2, Retinal (FSCN2), Radixin (RDX), POU Class 4 Homeobox 3 (POU4F3), TRIO and F-actin binding protein (TRIOBP), Taperin (TPRN), Xin Actin Binding Repeat Containing 2 (XIRP2), Atonal BHLH Transcription Factor 1 (ATOH1), Transcriptional repressor Growth Factor Independent 1 (GFI1), Cholinergic Nicotinic Receptor Alpha 9 Subunit (CHRNA9), Cholinergic Nicotinic Receptor Alpha 10 Subunit (CHRNA10), Calcium and Integrin Binding Family Member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), MKRN2 Opposite Strand (MKRN2OS), LIM Homeobox Protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain-Containing Cysteine-Rich Protein 1 (Glutaredoxin Domain Containing Cysteine-Rich Protein1) (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+ Transporting 2 (ATPase Plasma Membrane Ca2+ Transporting 2) 2) (ATP2B2), calcium and integrin binding family member 2 (CIB2), voltage-dependent calcium channel auxiliary subunit alpha 2 delta 4 (CACNA2D4), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), peripherin 2 (PRPH2), solute carrier family 8 member A2 (SLC8A2), zinc finger CCHC-type containing protein 12 (ZCCHC12), leucine rich transmembrane and O-methyltransferase domain containing The proteins involved in the expression of LRTOMT2 and LRTOMT1, USH1 protein network component harmonin (USH1C), solute carrier family 26 member 5 (SLC26A5), Piezo-type mechanosensitive ion channel component 2 (PIEZO2), and extracellular leucine rich repeat and fibronectin type III domain containing 1 (Extracellular Leucine Rich Repeat and Fibronectin Type III Domain Containing ...1 (ELFN1), Tetratricopeptide Repeat Protein 24 (TTC24), Dystrotelin (DYTN), Coiled-coil Glutamic Acid-rich Protein 2 (CCER2), Leucine-rich Repeat and Transmembrane Domain-containing Protein 2 (LRTM2), Voltage-gated Potassium Channel Subfamily A Member 10 (KCNA10), Clarin 1 (CLRN1), Clarin 2 (CLRN2), SKI Family Transcriptional Corepressor 1 (SKOR1), Tctex1 Domain-containing Protein 1 (TCTEX1D1), Fc Receptor-like B (FCRLB), Glutaredoxin Domain-containing Cysteine-rich Protein 2 (GRXCR2), Serpin Family E Member 3 (SERPINE3), Nescient Helix-loop Helix 1 (Nescient Helix-loop Helix 1) (NHLH1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), activating transcription factor 6 (ATF6), eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), serine / threonine protein kinase / endoribonuclease IRE1 (IRE1), Whirlin (WHRN), oncomodulin (OCM), LIM homeobox 1 (Isl1), transmembrane and tetratricopeptide repeat-containing 4 (TMTC4), binding immunoglobulin protein (BIP), or voltage-gated potassium channel subfamily Q member 4 (KCNQ4).

[0016] In some embodiments of any of the foregoing aspects, a linking polynucleotide is used to link the 3' end of the STRC promoter and the 5' start site (ATG) of the protein-encoding polynucleotide. In some embodiments, the linking polynucleotide comprises a Kozak sequence or a portion thereof. In some embodiments, the linking polynucleotide comprises a multiple cloning site or a portion thereof.

[0017] In some embodiments of any of the above aspects, the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, an adenoviral vector, or a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector has a capsid of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S. In some embodiments, the AAV vector has an AAV1 capsid. In some embodiments, the AAV vector has an AAV9 capsid. In some embodiments, the AAV vector has a 7m8 capsid. In some embodiments, the AAV vector has a PHP.S capsid. In some embodiments, the AAV vector has an Anc80 capsid. In some embodiments, the AAV vector has an Anc80L65 capsid. In some embodiments, the AAV vector has an AAV2 capsid. In some embodiments, the AAV vector has an AAV2quad(YF) capsid. In some embodiments, the AAV vector has a PHP.eB capsid. In some embodiments, the AAV vector has an AAV3 capsid. In some embodiments, the AAV vector has an AAV4 capsid. In some embodiments, the AAV vector has an AAV5 capsid. In some embodiments, the AAV vector has an AAV6 capsid. In some embodiments, the AAV vector has an AAV7 capsid. In some embodiments, the AAV vector has an AAV8 capsid. In some embodiments, the AAV vector has a PHP.B capsid.

[0018] In another aspect, the invention provides a nucleic acid vector comprising a STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2, operably linked to a first polynucleotide that does not encode a full-length stereocillin protein but encodes an N-terminal portion of a stereocillin protein. In some embodiments, the nucleic acid vector is a first nucleic acid vector in a two-vector system, further comprising a second nucleic acid vector containing a second polynucleotide encoding a C-terminal portion of a stereocillin protein.

[0019] In another aspect, the present invention provides a nucleic acid vector comprising a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 48 or a functional portion thereof including nucleotides 280-560 of SEQ ID NO: 48, operably linked to a first polynucleotide encoding an N-terminal portion of a stereocillin protein without encoding a full-length stereocillin protein. In some embodiments, the nucleic acid vector is a first nucleic acid vector in a two-vector system, further comprising a second nucleic acid vector containing a second polynucleotide encoding a C-terminal portion of a stereocillin protein.

[0020] In another aspect, the invention provides a method for the preparation of a stereocillin protein comprising: (a) a first polynucleotide encoding an N-terminal portion of a stereocillin protein; (i) a first polynucleotide encoding an N-terminal portion of a stereocillin protein; (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; or (ii) a second polynucleotide encoding an N-terminal portion of a stereocillin protein; operably linked to a first polynucleotide encoding an N-terminal portion of a stereocillin protein; The present invention provides a two-vector system that includes (a) a first nucleic acid vector containing a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion thereof, including 0; and (b) a second nucleic acid vector containing a second polynucleotide encoding a C-terminal portion of a stereocillin protein.

[0021] In another aspect, the invention provides a two-vector system including: (a) a first nucleic acid vector containing a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48 or a functional portion thereof including nucleotides 280-560 of SEQ ID NO:48 operably linked to a first polynucleotide encoding an N-terminal portion of a stereocillin protein; and (b) a second nucleic acid vector containing a second polynucleotide encoding a C-terminal portion of a stereocillin protein.

[0022] In some embodiments, the first polynucleotide overlaps with the second polynucleotide. In some embodiments, the first polynucleotide and the second polynucleotide have an overlapping region that is at least 200 bases (b) in length (e.g., at least 200b, 300b, 400b, 500b, 600b, 700b, 800b, 900b, 1.0 kilobase (kb), 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb or more). In some embodiments, the first nucleic acid vector and the second nucleic acid vector undergo homologous recombination when introduced into a mammalian cell to form a recombinant polynucleotide that encodes a full-length stereocillin protein.

[0023] In some embodiments, the first nucleic acid vector includes a splice donor signal sequence located 3' to the first polynucleotide and the second nucleic acid vector includes a splice acceptor signal sequence located 5' to the second polynucleotide. In some embodiments, the first polynucleotide and the second polynucleotide do not overlap.

[0024] In some embodiments, the first nucleic acid vector includes a splice donor signal sequence located 3' of the first polynucleotide and a first recombination induction region located 3' of the splice donor signal sequence, and the second nucleic acid vector includes a second recombination induction region, a splice acceptor signal sequence located 3' of the recombination induction region, and a second polynucleotide located 3' of the splice acceptor signal sequence. In some embodiments, the first polynucleotide and the second polynucleotide do not overlap. In some embodiments, the first recombination induction region and the second recombination induction region are the same. In some embodiments, each of the first recombination induction region and the second recombination induction region is an AP gene fragment. In some embodiments, the AP gene fragment includes or consists of any one of SEQ ID NOs: 42-47. In some embodiments, the AP gene fragment includes or consists of SEQ ID NO: 45. In some embodiments, the first nucleic acid vector further comprises a degradation signal sequence located 3' of the recombinogenic region and the second nucleic acid vector further comprises a degradation signal sequence located between the recombinogenic region and the splice acceptor signal sequence.

[0025] In some embodiments, the second nucleic acid vector further comprises a STRC promoter operably linked to the second polynucleotide, the STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2, wherein the STRC promoter is located 5' to the second polynucleotide.

[0026] In some embodiments, the second nucleic acid vector further comprises a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48 or a functional portion thereof including nucleotides 280-560 of SEQ ID NO:48 operably linked to the second polynucleotide, wherein the STRC promoter is located 5' to the second polynucleotide.

[0027] In some embodiments of any of the preceding aspects, the STRC promoter in the second nucleic acid vector is the same (i.e., has the same nucleotide sequence) as the STRC promoter in the first nucleic acid vector. In some embodiments of any of the preceding aspects, the STRC promoter in the second nucleic acid vector has a different nucleotide sequence than the STRC promoter in the first nucleic acid vector.

[0028] In some embodiments of any of the aforementioned aspects, the STRC promoter has at least 85% sequence identity to SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0029] In some embodiments of any of the aforementioned aspects, the STRC promoter consists of SEQ ID NO:1. In some embodiments of any of the foregoing aspects, the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof including nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0030] In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 252-537 of SEQ ID NO:2. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 120-537 of SEQ ID NO:2. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:2 includes or consists of nucleotides 35-530 of SEQ ID NO:2.

[0031] In some embodiments of any of the aforementioned aspects, the STRC promoter consists of SEQ ID NO:2. In some embodiments of any of the aforementioned aspects, the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48.

[0032] In some embodiments of any of the aforementioned aspects, the STRC promoter has the sequence of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 280-560 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 280-564 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 124-560 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 124-564 of SEQ ID NO:48. In some embodiments of any of the preceding aspects, the functional portion of SEQ ID NO:48 includes or consists of nucleotides 1-560 of SEQ ID NO:48.

[0033] In some embodiments of any of the preceding aspects, the first nucleic acid vector further includes a polynucleotide encoding an N-terminal intein (N-intein) located 3' to and in reading frame with the first polynucleotide. In some embodiments of any of the preceding aspects, the second nucleic acid vector further includes a polynucleotide encoding a C-terminal intein (C-intein) located between the STRC promoter and the second polynucleotide and in reading frame with the second polynucleotide. In some embodiments, the N-intein and the C-intein are components of a split intein trans-splicing system.

[0034] In some embodiments of any of the foregoing aspects, the first and / or second vector comprises an intein degradation signal. In some embodiments, the degradation signal is an N-degron and / or a C-degron. In some embodiments, the N-degron and / or the C-degron are, independently, a CL1 degron, a PB29 degron, an SMN degron, a CIITA degron, or an ODC degron. In some embodiments, the degradation signal is an E. coli dihydrofolate reductase (ecDHFR) degradation signal. In some embodiments, the degradation signal is an FKBP12 degradation domain (Banaszynski et al., Cell 126:995-1004, 2006). In some embodiments, the degradation signal is a PEST degradation domain (Rechsteiner and Rogers, Trends Biochem Sci. 21:267-271, 1996). In some embodiments, the degradation signal is a UbR tag ubiquitination signal (Chassin et al., Nat Commun. 10:2013, 2019). In some embodiments, the degradation signal is a destabilizing mutation of human ELRBD (Miyazaki et al., J. Am. Chem. Soc., 134:3942-3945, 2012).

[0035] In some embodiments of any of the foregoing aspects, the first vector and the second vector, when introduced into a mammalian (e.g., human) hair cell (e.g., an inner hair cell, an outer hair cell, a type I vestibular hair cell, or a type II vestibular hair cell), produce a first fusion protein and a second fusion protein, respectively, where the first fusion protein includes an N-terminal portion of a stereocillin protein and an N-intein located 3' thereto, and the second fusion protein includes a C-intein and a C-terminal portion of a stereocillin protein located 3' thereto. In some embodiments, the C-terminus of the N-intein of the first fusion protein and the N-terminus of the C-intein of the second fusion protein can form a peptide bond to generate a polypeptide that includes, from the N-terminus to the C-terminus, the N-terminal portion of a stereocilin protein, the N-intein, the C-intein, and the C-terminal portion of a stereocilin protein, and the combined N-intein and C-intein can excise themselves to link the C-terminus of the N-terminal portion of the stereocilin protein to the N-terminus of the C-terminal portion of the stereocilin protein, thereby generating a full-length stereocilin protein.

[0036] In some embodiments of any of the aforementioned aspects, the split intein trans-splicing system is derived from one or more bacterial DnaE genes. In some embodiments, the one or more bacteria are Nostoc punctiforme (Npu), Synechocystis sp. PCC6803 (Ssp), Fischerella sp. flagelliforme(Nfl), Crocosphaera watsonii(Cwa)WH8502, Chroococcidiopsis cubana(Ccu)CCALA043, Trichodesmium erythraeum(Ter), Rhodothermus marinus(Rma), Saccharomyces cerevisiae(Sce), Saccharomyces castellii(Sca), Saccharomyces unisporus (Sun), Zygosaccharomyces bisporus (Zbi), Torulaspora pretoriensis (Tpr), Mycobacteria tuberculosis (Mtu), Mycobacterium leprae (Mle), Mycobacterium smegmatis (Msm), Pyrococcus abyssi (Pab), Pyrococcus horikoshii (Pho), Coxiella burnetti (Cbu), Coxiella neoformans (Cne), Coxiella gattii (Cga), Histoplasma capsulatum (Hca), and Porphyra purpurea chloroplast (Ppu). In some embodiments, the split intein trans-splicing system is derived from multiple sequence alignment studies of DnaE to identify a consensus design (e.g., Cfa) for engineering a split intein with desired stability and activity.

[0037] In some embodiments of any of the foregoing aspects, the N-intein has the sequence of any one of SEQ ID NOs: 7, 9, 12, 14, 16-21, 26, 28, 30, 32, 34, 36, 38, 49, 51, 53, 55, and 57, and the C-intein has the sequence of any one of SEQ ID NOs: 8, 10, 11, 13, 15, 22-25, 27, 29, 31, 33, 35, 37, 39, 50, 52, 54, 56, and 58. In some embodiments, the N-intein has the sequence of SEQ ID NO: 7, and the C-intein has the sequence of SEQ ID NO: 8. In some embodiments, the N-intein has the sequence of SEQ ID NO: 7, and the C-intein has the sequence of SEQ ID NO: 10. In some embodiments, the N-intein has the sequence of SEQ ID NO: 7, and the C-intein has the sequence of SEQ ID NO: 11. In some embodiments, the N-intein has the sequence of SEQ ID NO: 9, and the C-intein has the sequence of SEQ ID NO: 8. In some embodiments, the N-intein has a sequence of SEQ ID NO:9 and the C-intein has a sequence of SEQ ID NO:10. In some embodiments, the N-intein has a sequence of SEQ ID NO:9 and the C-intein has a sequence of SEQ ID NO:11. In some embodiments, the N-intein has a sequence of SEQ ID NO:12 and the C-intein has a sequence of SEQ ID NO:13. In some embodiments, the N-intein has a sequence of SEQ ID NO:14 and the C-intein has a sequence of SEQ ID NO:15. In some embodiments, the N-intein has a sequence of SEQ ID NO:16 and the C-intein has a sequence of SEQ ID NO:22. In some embodiments, the N-intein has a sequence of SEQ ID NO:19 and the C-intein has a sequence of SEQ ID NO:23. In some embodiments, the N-intein has a sequence of SEQ ID NO:20 and the C-intein has a sequence of SEQ ID NO:24. In some embodiments, the N-intein has a sequence of SEQ ID NO:21 and the C-intein has a sequence of SEQ ID NO:25. In some embodiments, the N-intein has a sequence of SEQ ID NO:26 and the C-intein has a sequence of SEQ ID NO:27. In some embodiments, the N-intein has the sequence of SEQ ID NO:28 and the C-intein has the sequence of SEQ ID NO:29.In some embodiments, the N-intein has a sequence of SEQ ID NO: 30 and the C-intein has a sequence of SEQ ID NO: 31. In some embodiments, the N-intein has a sequence of SEQ ID NO: 32 and the C-intein has a sequence of SEQ ID NO: 33. In some embodiments, the N-intein has a sequence of SEQ ID NO: 34 and the C-intein has a sequence of SEQ ID NO: 35. In some embodiments, the N-intein has a sequence of SEQ ID NO: 36 and the C-intein has a sequence of SEQ ID NO: 37. In some embodiments, the N-intein has a sequence of SEQ ID NO: 38 and the C-intein has a sequence of SEQ ID NO: 39. In some embodiments, the N-intein has a sequence of any one of SEQ ID NOs: 16-21 and the C-intein has a sequence of any one of SEQ ID NOs: 22-25. In some embodiments, the N-intein has a sequence of SEQ ID NO: 49 and the C-intein has a sequence of SEQ ID NO: 50. In some embodiments, the N-intein has a sequence of SEQ ID NO: 51 and the C-intein has a sequence of SEQ ID NO: 52. In some embodiments, the N-intein has a sequence of SEQ ID NO: 53 and the C-intein has a sequence of SEQ ID NO: 54. In some embodiments, the N-intein has a sequence of SEQ ID NO: 55 and the C-intein has a sequence of SEQ ID NO: 56. In some embodiments, the N-intein has a sequence of SEQ ID NO: 57 and the C-intein has a sequence of SEQ ID NO: 58. In some embodiments, the split intein trans-splicing system includes one or more inteins that perform trans-splicing of a protein only upon contact with a ligand. In some embodiments, the ligand is selected from the group consisting of 4-hydroxytamoxifen, a peptide, a protein, a polynucleotide, an amino acid, and a nucleotide.

[0038] In some embodiments of any of the foregoing aspects, a linking polynucleotide is used to link the 3' end of the STRC promoter and the 5' start site (ATG) of the first polynucleotide and / or the polynucleotide encoding the C-intein. In some embodiments, the linking polynucleotide includes a Kozak sequence or a portion thereof. In some embodiments, the linking polynucleotide includes a multiple cloning site or a portion thereof.

[0039] In some embodiments of any of the preceding aspects, the first nucleic acid vector further comprises a polynucleotide encoding a signal peptide. In some embodiments, the polynucleotide encoding the signal peptide is positioned 5' and in frame with the polynucleotide encoding the N-terminal portion of the stereocillin protein. In some embodiments of any of the preceding aspects, the second nucleic acid vector further comprises a polynucleotide encoding a signal peptide. In some embodiments, the polynucleotide encoding the signal peptide is positioned 5' and in frame with the polynucleotide encoding the C-terminal portion of the stereocillin protein.

[0040] In some embodiments of any of the preceding aspects, neither the first polynucleotide nor the second polynucleotide encodes a full-length stereocillin protein. In some embodiments of any of the preceding aspects, each of the first polynucleotide and the second polynucleotide encodes about half of the stereocillin protein sequence.

[0041] In some embodiments of any of the aforementioned aspects, the second nucleic acid vector further comprises a poly(A) sequence 3' to the second polynucleotide. In some embodiments of any of the preceding aspects, the first nucleic acid vector and the second nucleic acid vector do not include a STRC untranslated region (UTR) that is not part of a promoter described herein. In some embodiments of any of the preceding aspects, the first nucleic acid vector and the second nucleic acid vector include a STRC UTR. In some embodiments of any of the preceding aspects, the first nucleic acid vector includes a 5' STRC UTR 5' of the first polynucleotide. In some embodiments of any of the preceding aspects, the second nucleic acid vector includes a 3' STRC UTR 3' of the second polynucleotide.

[0042] In some embodiments of any of the preceding aspects, the first and second polynucleotides encoding portions of the stereocillin protein do not include an intron (e.g., the first and second polynucleotides are portions of a STRC cDNA). In some embodiments of any of the preceding aspects, the first and second polynucleotides encoding portions of the stereocillin protein include an intron.

[0043] In some embodiments of any of the foregoing aspects, the two-vector system can direct hair cell-specific expression of full-length stereocillin protein to mammalian hair cells. In some embodiments, the mammalian hair cells are human hair cells. In some embodiments, the mammalian hair cells are mouse hair cells. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the cochlear hair cells are outer hair cells. In some embodiments, the cochlear hair cells are inner hair cells. In some embodiments, the hair cells are vestibular hair cells. In some embodiments, the vestibular hair cells are type I vestibular hair cells. In some embodiments, the vestibular hair cells are type II vestibular hair cells.

[0044] In some embodiments of any of the foregoing aspects, the stereocillin protein is a human stereocillin protein having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3. In some embodiments, the stereocillin protein has the sequence of SEQ ID NO:3. In some embodiments, the human stereocillin protein is encoded by a polynucleotide having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, a polynucleotide having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5 encodes the stereocillin protein of SEQ ID NO: 3. In some embodiments, the human stereocillin protein is encoded by a polynucleotide having the sequence of SEQ ID NO:5.

[0045] In some embodiments, the stereocillin protein is a mouse stereocillin protein having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4. In some embodiments, the stereocillin protein has the sequence of SEQ ID NO:4. In some embodiments, the mouse stereocillin protein is encoded by a polynucleotide having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6. In some embodiments, a polynucleotide having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6 encodes the stereocillin protein of SEQ ID NO: 4. In some embodiments, the mouse stereocillin protein is encoded by a polynucleotide having the sequence of SEQ ID NO:6.

[0046] In some embodiments of any of the above aspects, the first vector and the second vector are viral vectors, plasmids, cosmids, or artificial chromosomes. In some embodiments, the first vector and the second vector are viral vectors. In some embodiments, the viral vector is an AAV vector, an adenoviral vector, or a lentiviral vector. In some embodiments, the first vector and the second vector are AAV vectors. In some embodiments, each of the first AAV vector and the second AAV vector has a capsid of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S. In some embodiments, the first AAV vector and the second AAV vector each have an AAV1 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV9 capsid. In some embodiments, the first AAV vector and the second AAV vector each have a 7m8 capsid. In some embodiments, the first AAV vector and the second AAV vector each have a PHP.S capsid. In some embodiments, the first AAV vector and the second AAV vector each have an Anc80 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an Anc80L65 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV2 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV2quad(YF) capsid. In some embodiments, the first AAV vector and the second AAV vector each have a PHP.eB capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV3 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV4 capsid.In some embodiments, the first AAV vector and the second AAV vector each have an AAV5 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV6 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV7 capsid. In some embodiments, the first AAV vector and the second AAV vector each have an AAV8 capsid. In some embodiments, the first AAV vector and the second AAV vector each have a PHP.B capsid.

[0047] In another aspect, the present invention provides a composition comprising a nucleic acid vector or two-vector system according to any of the preceding aspects or embodiments. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0048] In another aspect, the present invention provides a cell containing a polynucleotide, a nucleic acid vector, or a two-vector system according to any of the preceding aspects or embodiments. In some embodiments, the cell is a hair cell. In some embodiments, the hair cell is a mammalian hair cell. In some embodiments, the mammalian hair cell is a human hair cell. In some embodiments, the hair cell is a cochlear hair cell. In some embodiments, the cochlear hair cell is an outer hair cell. In some embodiments, the cochlear hair cell is an inner hair cell. In some embodiments, the hair cell is a vestibular hair cell. In some embodiments, the vestibular hair cell is a type II vestibular hair cell. In some embodiments, the vestibular hair cell is a type I vestibular hair cell.

[0049] In another aspect, the invention provides a method of expressing a heterologous expression product in a hair cell by contacting the hair cell with a nucleic acid vector or composition according to any of the preceding aspects or embodiments. In some embodiments, the contacting is performed in vivo (e.g., within a subject). In some embodiments, the expression product is specifically expressed in the hair cell.

[0050] In another aspect, the present invention provides a method for expressing a stereocillin protein in a hair cell by contacting the hair cell with a two-vector system or composition according to any of the preceding aspects or embodiments. In some embodiments, the contacting is performed in vivo (e.g., within a subject). In some embodiments, the stereocillin protein is specifically expressed in hair cells.

[0051] In another aspect, the invention provides a method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss, nonsyndromic hearing loss, auditory neuropathy, or hearing loss) by administering to the subject's inner ear an effective amount of a nucleic acid vector, two-vector system, or composition described in any of the preceding aspects or embodiments.

[0052] In another aspect, the invention provides a method of treating a subject having or at risk of developing tinnitus by administering to the subject's inner ear an effective amount of a nucleic acid vector or composition as described in any of the preceding aspects or embodiments.

[0053] In another aspect, the invention provides a method of treating a subject having or at risk of developing a vestibular dysfunction by administering to the subject's inner ear an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments.

[0054] In another aspect, the invention provides a method of treating a subject having or at risk of developing a bilateral vestibular disorder by administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments. In some embodiments, the bilateral vestibular disorder is an ototoxic drug-induced bilateral vestibular disorder.

[0055] In another aspect, the present invention provides a method of treating a subject having or at risk of developing oscillopsia by administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments. In some embodiments, the oscillopsia is ototoxic drug-induced oscillopsia.

[0056] In another aspect, the invention provides a method of treating a subject having or at risk of developing a balance disorder by administering to the subject's inner ear an effective amount of a nucleic acid vector, two-vector system, or composition described in any of the preceding aspects or embodiments.

[0057] In another aspect, the invention provides a method of inducing or increasing hair cell regeneration in a subject in need thereof by administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition according to any of the preceding aspects or embodiments.

[0058] In another aspect, the invention provides a method of increasing hair cell maintenance in a subject in need thereof by administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments.

[0059] In another aspect, the invention provides a method of increasing hair cell survival in a subject in need thereof by administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments.

[0060] In another aspect, the invention provides a method of inducing or increasing hair cell maturation in a subject in need thereof by administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition according to any of the preceding aspects or embodiments.

[0061] In another aspect, the invention provides a method of preventing or reducing ototoxic drug-induced hair cell damage or cell death in a subject in need thereof by administering to the inner ear of a subject an effective amount of a nucleic acid vector, two-vector system, or composition as described in any of the preceding aspects or embodiments.

[0062] In another aspect, the invention provides a method of preventing or reducing hair cell damage or cell death in a subject in need thereof by administering to the inner ear of a subject an effective amount of a nucleic acid vector, two-vector system, or composition as described in any of the preceding aspects or embodiments.

[0063] In another aspect, the invention provides a method of improving hair cell function in a subject in need thereof by administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition according to any of the preceding aspects or embodiments.

[0064] In another aspect, the invention provides a method of increasing or improving hair bundle adhesion (e.g., hair bundle adhesion of OHCs) to the tectorial membrane in a subject in need thereof, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector, two-vector system, or composition described in any of the preceding aspects or embodiments.

[0065] In another aspect, the invention provides a method of increasing expression of STRC (e.g., expression of wild-type STRC, e.g., to produce wild-type stereocillin protein) in a subject in need thereof (e.g., hair cells of a subject) by administering to the inner ear of a subject a therapeutically effective amount of a two-vector system or composition as described in any of the preceding aspects or embodiments.

[0066] In some embodiments of any of the preceding aspects, the hair cells are mammalian hair cells. In some embodiments, the mammalian hair cells are human hair cells. In some embodiments of any of the preceding aspects, the hair cells are hair cells that endogenously express STRC. In some embodiments of any of the preceding aspects, the hair cells are cochlear hair cells. In some embodiments, the cochlear hair cells are outer hair cells. In some embodiments, the cochlear hair cells are inner hair cells. In some embodiments of any of the preceding aspects, the hair cells are vestibular hair cells. In some embodiments, the vestibular hair cells are type II vestibular hair cells. In some embodiments, the vestibular hair cells are type I vestibular hair cells.

[0067] In some embodiments of any of the preceding aspects, the subject has or is at risk of developing hearing loss (e.g., nonsyndromic hearing loss, auditory neuropathy, or sensorineural hearing loss such as hearing loss). In some embodiments of any of the preceding aspects, the hearing loss is genetic hearing loss. In some embodiments, the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss. In some embodiments of any of the preceding aspects, the hearing loss is acquired hearing loss. In some embodiments, the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

[0068] In some embodiments of any of the above aspects, the subject has vestibular dysfunction or is at risk of developing vestibular dysfunction. In some embodiments of any of the above aspects, the vestibular dysfunction is vertigo, dizziness, imbalance, bilateral vestibular dysfunction, oscillopsia, or balance disorder. In some embodiments of any of the above aspects, the vestibular dysfunction is age-related vestibular dysfunction, head trauma-related vestibular dysfunction, disease or infection-related vestibular dysfunction, or ototoxic drug-induced vestibular dysfunction. In some embodiments of any of the above aspects, the vestibular dysfunction is associated with a genetic mutation. In some embodiments of any of the above aspects, the vestibular dysfunction is idiopathic vestibular dysfunction.

[0069] In some embodiments of any of the foregoing aspects, the ototoxic drug is an aminoglycoside (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, or amikacin), antineoplastic agent (e.g., platinum-containing chemotherapy agents such as cisplatin, carboplatin, and oxaliplatin), ethacrynic acid, furosemide, a salicylate (e.g., aspirin, especially at high doses), or quinine.

[0070] In some embodiments of any of the preceding aspects, the hearing loss, vestibular dysfunction, or tinnitus is associated with hair cell loss, hair cell damage, or dysfunction of hair cells (e.g., cochlear and / or vestibular hair cells). In some embodiments of any of the preceding aspects, the hearing loss or vestibular dysfunction is associated with abnormal tilt of hair cell stereocilia bundles or abnormal connectivity between hair bundles (e.g., hair bundles of OHCs) and the tectorial membrane.

[0071] In some embodiments of any of the above aspects, the subject has a mutation in STRC. In some embodiments of any of the above aspects, the subject has been identified as having a mutation in STRC. In some embodiments of any of the above aspects, the method further includes identifying the subject as having a mutation in STRC before administering the two-vector system or pharmaceutical composition. In some embodiments of any of the above aspects, the subject has hearing loss, autosomal recessive 16 (DFNB16). In some embodiments of any of the above aspects, the subject has been identified as having DFNB16.

[0072] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's hearing (e.g., assessing hearing using a standard test such as a hearing test, auditory brainstem response (ABR), electrocochleogram (ECOG), or otoacoustic emissions) prior to administration of the nucleic acid vector, two-vector system, or composition.

[0073] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's hearing (e.g., assessing hearing using a standard test such as audiometry, ABR, ECOG, or otoacoustic emissions) after administration of the nucleic acid vector, two-vector system, or composition.

[0074] In some embodiments of any of the foregoing aspects, the method further includes assessing the subject's vestibular function prior to administration of the nucleic acid vector, two-vector system, or composition (e.g., using standard tests such as an electronystagmogram (ENG) or video nystagmography (VNG), a test of the vestibulo-ocular reflex (VOR) (e.g., a head impulse test (Halmagyi-Curthoys test), which can be performed both at the bedside or using video head impulse testing (VHIT), or a caloric reflex test), postural sway tests, rotary-chair testing, ECOG, vestibular evoked myogenic potential testing (VEMP), or a specialized outpatient balance test such as that described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010)).

[0075] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's vestibular function after administration of the nucleic acid vector, two-vector system, or composition (e.g., assessing vestibular function using standard tests such as ENG, VNG, VOR testing, stabilometry, rotary-chair testing, ECOG, VEMP, or balance testing in a specialized clinic).

[0076] In some embodiments of any of the foregoing aspects, the nucleic acid vector, two-vector system, or composition is administered locally. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered to the inner ear. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered to the middle ear. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered to the semicircular canal. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered transtympanically or intratympanically. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered into the perilymph. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered into the endolymph. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered to or through the oval window. In some embodiments, the nucleic acid vector, two-vector system, or composition is administered to or through the round window.

[0077] In some embodiments of any of the aforementioned aspects, the vectors of the two-vector system are administered simultaneously. In some embodiments of any of the aforementioned aspects, the vectors of the two-vector system are administered sequentially.

[0078] In some embodiments of any of the foregoing aspects, the nucleic acid vector, two-vector system, or composition is capable of preventing or reducing vestibular dysfunction, delaying the onset of vestibular dysfunction, slowing the progression of vestibular dysfunction, improving vestibular function, preventing or reducing hearing loss, preventing or reducing tinnitus, delaying the onset of hearing loss, slowing the progression of hearing loss, improving hearing, improving speech discrimination, improving hair cell function, increasing expression of STRC in hair cells, increasing the expression of STRC in hair cells in the cochlea and / or vestibule. The compound is administered in an amount sufficient to increase cell number, enhance maturation of cochlear and / or vestibular hair cells, increase regeneration of cochlear and / or vestibular hair cells, improve cochlear and / or vestibular hair cell function, prevent or reduce damage to cochlear and / or vestibular hair cells, prevent or reduce cochlear and / or vestibular hair cell death, improve hair bundle attachment to the tectorial membrane (e.g., hair bundle attachment of OHCs), or promote or increase survival of cochlear and / or vestibular hair cells.

[0079] In some embodiments of any of the aforementioned aspects, the subject is a human subject. In another aspect, the invention provides a kit containing a polynucleotide, a nucleic acid vector, a two-vector system, or a composition according to any of the preceding aspects or embodiments.

[0080] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0081] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector containing a STRC promoter operably linked to a transgene) to a subject by any effective route. Exemplary administration routes are described herein below.

[0082] As used herein, the phrase "administering to the inner ear" refers to providing or giving a therapeutic agent described herein to a subject by any route that allows for transduction of inner ear cells. Exemplary routes of administration to the inner ear include administration into the perilymph or endolymph, e.g., into or through the oval window, round window, or semicircular canal (e.g., the horizontal semicircular canal), or by transtympanic or intratympanic injection, e.g., into hair cells.

[0083] As used herein, the term "cell type" refers to a group of cells sharing a phenotype that can be statistically separated based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional properties, such as similar gene activation patterns and antigen presentation properties. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial, nervous, connective, or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0084] As used herein, the term "cochlear hair cells" refers to a group of specialized cells in the inner ear that are involved in the perception of sound. There are two types of cochlear hair cells: inner hair cells and outer hair cells. Damage to cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are implicated in hearing loss and hearing loss.

[0085] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk, as summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0086] [Table 1]

[0087] From this table, it is recognized that conservative amino acid families include: (i) G, A, V, L, and I, (ii) D and E, (iii) C, S and T, (iv) H, K and R, (v) N and Q, and (vi) F, Y and W. Thus, a conservative mutation or substitution is one that replaces an amino acid with a member of the same amino acid family (e.g., replacing Thr with Ser or Arg with Lys).

[0088] As used herein, the term "degradation signal sequence" refers to a sequence (e.g., a nucleotide sequence that can be translated into an amino acid sequence) that mediates the degradation of a polypeptide that contains it. A degradation signal sequence can be included in the nucleic acid vector of the present disclosure to reduce or prevent expression of portions of the stereocillin protein that have not been recombined and / or spliced.

[0089] The terms "derived" and "derivative" as used herein refer to a nucleic acid, peptide, or protein, or a variant or analog thereof, that contains one or more mutations and / or chemical modifications compared to a corresponding full-length wild-type nucleic acid, peptide, or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or combinations thereof.

[0090] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a mammal, e.g., a human, and thus "effective amount" or its synonyms vary depending on the context in which it is applied. For example, in the context of treating sensorineural hearing loss or vestibular dysfunction, it is the amount of the composition, vector construct, or viral vector that is sufficient to achieve a therapeutic response compared to the response obtained in the absence of administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, characteristics of the subject or host being treated (e.g., age, sex, weight), etc., but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition, vector construct, or viral vector of the present disclosure can be readily determined by one of ordinary skill in the art by routine methods known in the art. Dosage regimens can be adjusted to obtain optimal therapeutic effects.

[0091] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or a human cell, e.g., a cell such as a human hair cell).

[0092] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein. The term "expression product" refers to a protein or RNA molecule produced by any of these events.

[0093] As used herein, the term "exogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell, e.g., a human hair cell). Exogenous materials include those provided from a source external to the organism or culture materials extracted therefrom.

[0094] As used herein, the term "exon" refers to a region in the coding region of a gene, the nucleotide sequence of which determines the amino acid sequence of the corresponding protein. The term exon also refers to the corresponding region of the RNA transcribed from the gene. Exons are transcribed into pre-mRNA and may be included in the mature mRNA depending on alternative splicing of the gene. The exons included in the mature mRNA after processing are translated into a protein, in which case the sequence of the exon determines the amino acid composition of the protein.

[0095] As used herein, the term "functional portion," when referring to a promoter sequence described herein (e.g., the STRC promoter sequence), refers to a nucleotide sequence that is shorter than a promoter sequence described in Table 2 (e.g., SEQ ID NO: 2 or SEQ ID NO: 48) and that is capable of recruiting RNA polymerase to drive transcription of a gene to which it is operably linked. For example, in the context of the present disclosure, a functional portion of the mouse STRC promoter of SEQ ID NO: 2 (537 bases (b)) may have the sequence of SEQ ID NO: 2 or may include nucleotides 252 to 537 thereof. Other functional portions of the STRC promoter of SEQ ID NO: 2 may have the sequence of SEQ ID NO: 2 or may include nucleotides 120 to 537 or nucleotides 35 to 530 thereof. In another example, a functional portion of the human STRC promoter of SEQ ID NO: 48 (564 bases (b)) may have the sequence of SEQ ID NO: 48 or may include nucleotides 280 to 560 thereof. Other functional portions of the STRC promoter of SEQ ID NO:48 may have the sequence of SEQ ID NO:48 or may include nucleotides 280 to 564, nucleotides 124 to 560, nucleotides 124 to 564, nucleotides 61 to 560 (as set forth in SEQ ID NO:1), or nucleotides 1 to 560 thereof.

[0096] As used herein, the term "heterologous" refers to a combination of elements that do not occur in nature. For example, a heterologous transgene refers to a transgene that is not naturally expressed by a promoter to which it is operably linked.

[0097] As used herein, the term "hair cell" refers to a specialized sensory cell of the inner ear that transmits auditory (i.e., cochlear hair cell) or vestibular (i.e., vestibular hair cell) information. Hair cells are characterized by a bundle of stereocilia emerging from the apical surface of the cell. Examples of auditory (i.e., cochlear) hair cells include inner hair cells (IHCs) and outer hair cells (OHCs). Examples of vestibular hair cells include type I vestibular hair cells and type II vestibular hair cells.

[0098] As used herein, the term "hair cell-specific expression" refers to the production of an RNA transcript or polypeptide primarily in hair cells (e.g., cochlear hair cells and / or vestibular hair cells) as compared to other cell types of the inner ear (e.g., spiral ganglion neurons, glia, or other inner ear cell types). Hair cell-specific expression of a transgene can be confirmed by comparing transgene expression (e.g., RNA or protein expression) between various cell types of the inner ear (e.g., hair cells vs. non-hair cells) using any standard technique (e.g., quantitative RT PCR, immunohistochemical staining, Western blot analysis, or measuring the fluorescence of a reporter (e.g., GFP) operably linked to a promoter). A hair cell specific promoter directs expression of a transgene to which it is operably linked (e.g., expression of RNA or protein) in hair cells that is at least 50% greater (e.g., 50%, 75%, 100%, 125%, 150%, 175%, 200% or more greater) compared to at least three (e.g., three, four, five, six, seven, eight, nine, ten, or more) of the following inner ear cell types: border cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row Deiters cells, second row Deiters cells, third row Deiters cells, Hensen cells, Claudius cells, inner sulcus cells, outer sulcus cells, spiral eminence cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, Schwann cells. A hair cell specific promoter does not necessarily direct expression in all hair cells, but directs expression in at least one of the following hair cell types: inner hair cells, outer hair cells, type I vestibular hair cells, or type II vestibular hair cells. The STRC promoters described herein (e.g., SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:48, and portions thereof) are hair cell specific promoters.

[0099] As used herein, the terms "increasing" and "decreasing" refer to modulating the amount of a function, expression, or activity of an indicator to be greater or less than a reference, respectively. For example, after administration of a composition according to the methods described herein, the amount of a marker of an indicator described herein (e.g., transgene expression) can be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more in a subject compared to the amount of the marker before administration. In general, the indicator is measured after administration, at a time when administration has had the described effect, for example, at least 1 week, 1 month, 3 months, or 6 months after the start of the treatment regimen.

[0100] As used herein, the term "intein", also referred to as "protein intron", refers to a portion of a protein that is typically 100-900 amino acid residues long and capable of self-excision and ligation of adjacent protein fragments ("exteins") by peptide bonds. Inteins are generated during protein splicing. The term "intein" encompasses four different classes of inteins, including maxi-inteins, mini-inteins, trans-splicing inteins, and alanine inteins. Maxi-inteins refer to the N- and C-terminal splicing regions of a protein that contain an endonuclease domain. The endonuclease domain, also known as "homing endonuclease genes" or "HEGs", refers to a class of endonucleases that are encoded as standalone genes within introns, as protein fusions with other proteins, or as self-splicing inteins. HEGs generally hydrolyze highly underrepresented, often targeted regions of DNA. When HEG hydrolyzes a DNA fragment, the gene encoding HEG typically incorporates itself into the cleavage site, thereby increasing its allele frequency. Mini-intein refers to an N-terminal splicing domain and a C-terminal splicing domain that lacks an endonuclease domain. Trans-splicing intein refers to an intein that is split into two or more domains that are further split into an N-terminus and a C-terminus. Alanine intein refers to an intein that has an alanine splicing junction rather than a cysteine ​​or serine. In some cases, the intein of a precursor protein originates from two genes, in which case the intein is named a split "intein."

[0101] As used herein, the term "intron" refers to a region within the coding region of a gene whose nucleotide sequence is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of RNA transcribed from a gene. Introns are transcribed into pre-mRNA but are removed during processing and are not included in the mature mRNA.

[0102] As used herein, "locally" or "local administration" refers to administration at a particular site of the body where a local, rather than a systemic, effect is intended. Examples of local administration are administration to the skin of a subject, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional, lymph node, intratumor, inner ear, and mucous membranes, where such administration is intended to have a local, rather than a systemic, effect.

[0103] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, where the molecules are positioned such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule of interest if the promoter controls the transcription of the transcribable polynucleotide molecule of interest in a cell. Furthermore, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other by a linker polynucleotide (e.g., an intervening non-coding polynucleotide) or without the presence of an intervening nucleotide.

[0104] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule that can transport another nucleic acid to which it is ligated. Certain plasmids can replicate autonomously in a host cell into which they are introduced (e.g., bacterial plasmids with a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Certain plasmids can direct the expression of genes to which they are operatively linked.

[0105] As used herein, the term "polynucleotide" refers to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules that contain nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, and the like, whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When polynucleotides are referred to in this application, it is understood that both DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complementary strand of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or to sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in a 5' to 3' orientation, unless otherwise indicated.

[0106] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of the transgene. "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in a reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity of nucleic acids or amino acids can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids that are scored as a perfect match by a sequence alignment program (e.g., BLAST) in an alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0107] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharma- ceutically acceptable excipients, diluents, and / or carriers, administered to a subject, such as a mammal (e.g., a human), to prevent, treat, or manage a particular disease or condition that afflicts or may afflict the subject.

[0108] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0109] As used herein, the term "recombinogenic region" refers to a region of homology that mediates recombination between two distinct sequences. As used herein, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a polynucleotide. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990), which is incorporated herein by reference.

[0110] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., from the placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0111] As used herein, the terms "stereocillin" and "STRC" (also known as DFNB16) refer to the protein encoded by the STRC gene and the gene encoding this protein, respectively. In humans, the STRC gene is tandemly duplicated, where the second copy contains a premature stop codon in exon 20, thereby giving rise to a STRC pseudogene. In the context of the present disclosure, STRC does not refer to the STRC pseudogene. Previous studies have identified mutations in the full-length copy of STRC in human patients with autosomal recessive nonsyndromic sensorineural hearing loss (Verpy et al., Nat. Genet. 29:345-9 (2001)). Expression of stereocillin protein is restricted to the stereocilia of hair bundles of hair cells. Stereocilin is believed to form the apical lateral connectors and tectorial adhesion crown required for normal function of the hearing apparatus (Avan et al., PNAS 116:25948-57 (2019); Verpy et al., J. Comp. Neurol. 519:194-210 (2011)). Stereocilin-deficient mice have been shown to exhibit abnormal hair cell bundles and hearing impairment with adhesion defects (Verpy et al., Nature 456:255-8 (2008)). The present disclosure provides a polynucleotide encoding a full-length stereocilin protein, which, when incorporated into the vector system described herein, can be used as a therapeutic agent to treat hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular disorders, oscillopsia, or balance disorders) in a subject in need thereof.The terms "stereocillin" and "STRC" also refer to variants of the wild-type stereocillin protein and the nucleic acid encoding same, respectively, such as variants having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or greater) to the amino acid sequence of the wild-type stereocillin protein (e.g., SEQ ID NO:3 or SEQ ID NO:4). The term also refers to a polynucleotide having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or greater) to a wild-type STRC gene nucleic acid sequence (e.g., SEQ ID NO:5 or SEQ ID NO:6), provided that the encoded stereocillin analog retains the therapeutic function of wild-type (WT) stereocillin.

[0112] As used herein, the term "STRC promoter" refers to a promoter sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or greater, to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2, such as a portion containing nucleotides 252 to 537 or 35 to 530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48, such as a portion containing nucleotides 280 to 560 of SEQ ID NO:48.

[0113] As used herein, the term "transcriptional regulatory element" refers to a polynucleotide that at least partially controls the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other polynucleotides (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0114] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection, and the like.

[0115] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, such as a human). The subject treated according to the methods described herein may have been diagnosed with hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular disorders, oscillopia, or balance disorders), or may be at risk for developing one or both of these conditions. Diagnosis may be performed by any method or technique known in the art. One of skill in the art will understand that the subject treated according to the present disclosure may have undergone standard testing, or may have been identified as at risk without testing due to the presence of one or more risk factors associated with a disease or condition.

[0116] As used herein, the terms "transduction" and "transducing" refer to a method of introducing a vector construct or a portion thereof into a cell. In the case where the vector construct is contained within a viral vector, such as an AAV vector, transduction refers to viral infection of a cell and the subsequent introduction and integration of the vector construct or a portion thereof into the cellular genome.

[0117] As used herein, "treatment" and "treating" in reference to a disease or condition refer to an approach to obtain a beneficial or desired result, e.g., a clinical result. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; attenuation of the extent of the disease or condition; a stabilized (i.e., not worsening) state of the disease, disorder, or condition; prevention of the spread of the disease or condition; a delay or slowing of the progression of the disease or condition; an improvement or palliation of the disease or condition; and remission (whether partial or complete). "Ameliorating" or "alleviating" a disease or condition means that the extent and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or extended compared to the extent or time course in the absence of treatment. "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0118] As used herein, the term "vector" refers to a nucleic acid vector, such as a DNA vector, such as a plasmid, cosmid, or artificial chromosome, an RNA vector, a virus, or any other suitable replicon (e.g., a viral vector). A wide variety of vectors have been developed to deliver polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements that are used, for example, for protein expression and / or integration of these polynucleotide sequences into the mammalian cell genome. Particular vectors that can be used to express the transgenes described herein include vectors that contain regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other vectors useful for expressing transgenes contain polynucleotide sequences that enhance the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for the selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0119] As used herein, the term "vestibular hair cells" refers to a type of specialized cell in the inner ear that is involved in sensing motion and contributes to balance and spatial orientation. There are two types of vestibular hair cells: type I hair cells and type II hair cells. Type I hair cells have nerve ending cups, have a fast voltage response, and code for dynamic motion. Type II hair cells have nerve ending buttons, have a slow voltage response, and code for slow or static motion. Vestibular hair cells are located in the end organs of the semicircular canals and otolithic organs of the inner ear. Vestibular hair cell damage and genetic mutations that disrupt vestibular hair cell function have been implicated in vestibular dysfunction, such as vertigo, dizziness, imbalance, bilateral vestibular hypofunction, oscillopsia, and balance disorders.

[0120] As used herein, the term "wild type" refers to the most frequent genotype for a particular gene in a given organism. [Brief description of the drawings]

[0121] [Figure 1]

[0046] Figure 1 is a map of plasmid P1208. [Diagram 2] 1 is a map of plasmid P1209. [Diagram 3] FIG. 1 is a series of photomicrographs from the organ of Corti of a neonatal mouse administered an AAV vector derived from plasmid P1209 expressing GFP under the control of the STRC promoter at nucleotides 35-530 of SEQ ID NO:2. Panels A and B show composite serial sections from different planes of the organ of Corti, from basal to apical, from left to right. Panels A and A' show staining for Myo7a. Panel A' is a higher magnification of the area indicated by the rectangle in panel A. Panels B and B' show staining for GFP. Panel B' is a higher magnification of the area indicated by the rectangle in panel B. Scale bars represent 100 μm for panels A and B and 50 μm for panels A' and B'. [Figure 4]FIG. 1 is a series of photomicrographs from the organ of Corti of a neonatal mouse administered an AAV vector derived from plasmid P1208 expressing GFP under the control of the STRC promoter of SEQ ID NO: 1. Panels A and B show composite serial sections from different planes of the organ of Corti, from basal to apical, left to right. Panels A, A', and A'' show staining for Myo7a. Panel A' is a higher magnification of the area indicated by the solid rectangle in panel A. Panel A'' is a higher magnification of the area indicated by the dashed square in panel A. Panels B, B', and B'' show staining for GFP. Panel B' is a higher magnification of the area indicated by the rectangle in panel B. Panel B'' is a higher magnification of the area indicated by the dashed square in panel B. Scale bars represent 100 μm for panels A and B and 50 μm for panels A', A'', B', and B''. [Diagram 5] FIG. 1 is a series of photomicrographs of composite sections of the vestibule of neonatal mice administered the same plasmid P1209-derived AAV vector expressing GFP under the control of the STRC promoter at nucleotides 35-530 of SEQ ID NO:2. Panels A, A', and A'' show staining for Pou4f3. Panels B, B', and B'' show staining for GFP. Panels C, C', and C'' show staining for Sox2. Panels A', B', and C' show high-powered magnifications of corresponding areas within the squares in panels A, B, and C. Panels A'', B'', and C'' show high-powered magnifications of corresponding areas within the dashed lines in panels A', B', and C'. Scale bars represent 100 μm for panels A, B, and C, and 25 μm for panels A', A'', B', B'', C', and C''. [Figure 6] 1 is a series of photomicrographs of composite sections of the organ of Corti showing inner and outer hair cells of an untreated adult mouse stained for actin (left panel) and native stereocillin (right panel). Scale bars represent 10 μm. [Figure 7]FIG. 1 is a series of photomicrographs from the organ of Corti of an adult mouse administered an AAV vector derived from plasmid P1209 expressing GFP under the control of the STRC promoter at nucleotides 35-530 of SEQ ID NO:2. Panels A and B show composite serial sections from different planes of the organ of Corti, from basal to apical, from left to right. Panels A and A' show staining for Myo7a. Panel A' is a higher magnification of the area indicated by the rectangle in panel A. Panels B and B' show staining for GFP. Panel B' is a higher magnification of the area indicated by the rectangle in panel B. Scale bars represent 100 μm for panels A and B and 50 μm for panels A' and B'. [Figure 8] FIG. 1 is a series of photomicrographs from the organ of Corti of an adult mouse administered an AAV vector derived from plasmid P1208 expressing GFP under the control of the STRC promoter of SEQ ID NO: 1. Panels A and B show composite serial sections from different planes of the organ of Corti, from basal to apical, left to right. Panels A, A', and A'' show staining for Myo7a. Panel A' is a higher magnification of the area indicated by the solid rectangle in panel A. Panel A'' is a higher magnification of the area indicated by the dashed square in panel A. Panels B, B', and B'' show staining for GFP. Panel B' is a higher magnification of the area indicated by the rectangle in panel B. Panel B'' is a higher magnification of the area indicated by the dashed square in panel B. Scale bars represent 100 μm for panels A and B and 50 μm for panels A', A'', B', and B''. [Figure 9] FIG. 1 is a series of photomicrographs from the organ of Corti of a non-human primate (Macaca fascicularis) administered an AAV vector derived from plasmid P1016 expressing H2B-GFP under the control of the STRC promoter of SEQ ID NO: 1. Panels A and B show composite serial sections from different planes in the middle turn of the organ of Corti. Panel A shows staining of inner and outer hair cells. Panel B shows antibody staining for GFP. Scale bar represents 50 μm. Inner hair cells (IHC) and outer hair cells (OHC) are highlighted for orientation. [Figure 10]1. Microscopic images of a single paraffin section from the cochlear mid-turn of a non-human primate (Macaca fascicularis) administered an AAV vector derived from plasmid P1016 expressing H2B-GFP under the control of the STRC promoter of SEQ ID NO: 1. Panel A shows greyscale conversion of the pericoronal region with nuclei originally stained with hematoxylin in blue and H2B-GFP antibody originally stained in red. Panel B shows the remaining signal after removal of the blue hematoxylin signal; H2B-GFP positive (red) nuclei continue to be visible as darker colors after greyscale conversion. Scale bar represents 100 μm. Inner hair cells (IHCs) and outer hair cells (OHCs) are highlighted for localization. [Figure 11] 1 is a plasmid map of plasmid P1016. [Figure 12] A series of fluorescent images of stereocillin expression in the mouse organ of Corti in a 200 μm2 ROI at 16 kHz. A shows stereocillin antibody staining in the tips of the stereocilia of outer hair cells (OHCs) of wild-type CBA / CaJ mice. As shown in B, 232 bp STRC knockout (KO) animals lacked antibody signal. C shows stereocillin antibody staining in 232 bp STRC KO mice administered dual Anc80 vectors, the first vector carrying a CMV promoter and nucleotides 1-3200 of the mouse STRC cDNA, and the second vector carrying nucleotides 2201-5430, resulting in a 1000 bp overlap between the two cDNAs of the two vectors. De novo expression of stereocillin protein could be observed in the tips of OHC stereocilia and inner hair cell bodies in the organ of Corti of treated 232 bp STRC KO mice. [Figure 13A]13A-B are a series of graphs showing improved hearing function in 232bp STRC KO mice treated with Anc80-CMV-mStrc and correlation with STRC expression in OHCs. Untreated contralateral ears showed a near absence of DPOAEs and highly elevated ABR thresholds (FIGS. 13A and 13B, open circles), indicative of loss of OHC function, whereas treated 232bp STRC KO animals showed recovery of hearing thresholds (FIGS. 13A and 13B, filled circles). The best responders of treated animals (FIGS. 13A and 13B, filled squares) showed hearing thresholds close to those of wild type (FIGS. 13A and 13B, triangles). [Figure 13B] 13A-B are a series of graphs showing improved hearing function in 232bp STRC KO mice treated with Anc80-CMV-mStrc and correlation with STRC expression in OHCs. Untreated contralateral ears showed a near absence of DPOAEs and highly elevated ABR thresholds (FIGS. 13A and 13B, open circles), indicative of loss of OHC function, whereas treated 232bp STRC KO animals showed recovery of hearing thresholds (FIGS. 13A and 13B, filled circles). The best responders of treated animals (FIGS. 13A and 13B, filled squares) showed hearing thresholds close to those of wild type (FIGS. 13A and 13B, triangles). [Figure 13C]

[0023] Figure 1 is a series of graphs showing improved hearing function in 232bp STRC KO mice treated with Anc80-CMV-mStrc and correlation with STRC expression in OHCs. A higher percentage of OHCs in 232bp STRC KO mice expressing stereocillin after treatment with AAV-Anc80-CMV-mStrc was found to promote hearing recovery. [Figure 14] 1 shows images and graphs showing that transfection of the two-vector split-intein system into HEK293T cells resulted in reconstitution of full-length stereocillin. A is a representative image of a Western blot for stereocillin protein and beta-actin. B is a densitometric quantification of full-length stereocillin band intensity compared to actin, showing the relative expression of full-length stereocillin protein for the negative (GFP) control, the positive full-length control, and the Npu intein construct. [Figure 15] Graph showing the amount of stereocillin protein expression detected using a single plasmid vector or an AAV dual vector system in HEK293T cells. The full-length control was a plasmid DNA containing the full-length mouse STRC coding sequence. GFP was also expressed using a plasmid. The AAV dual hybrid vectors tested differed in the split site used to separate the mouse STRC sequence between the first and second vectors. The value on the x-axis indicates the last nucleotide of mouse STRC in the 5' vector (e.g., dual hybrid 1800 is a dual hybrid vector system in which the 5' vector contains nucleotides 1-1800 of mouse STRC and the remaining nucleotides of the STRC coding sequence starting at nucleotide 1801 are contained in the 3' vector). The overlap was an overlapping dual AAV vector system in which the 5' and 3' vectors shared a common 1,000 nucleotides from the STRC coding sequence (the 5' vector carried nucleotides 79 to 3278 of NM_080459 (corresponding to nucleotides 1 to 3200 of SEQ ID NO:6) and the 3' vector carried nucleotides 2279 to 5508 (corresponding to nucleotides 2201 to 5430 of SEQ ID NO:6)). [Figure 16A] A series of photomicrographs taken from mouse neonatal cochlear explants infected with AAV vectors expressing hypersensitive GFP under the control of various STRC promoters. The top row shows staining for the hair cell marker Myo7a and GFP. The middle row shows staining for Myo7a only. The bottom row shows staining for GFP only. Control photomicrographs were taken from untreated (non-AAV infected) explants. [Figure 16B] A series of photomicrographs taken from mouse neonatal cochlear explants infected with AAV vectors expressing hypersensitive GFP under the control of various STRC promoters. The top row shows staining for the hair cell marker Myo7a and GFP. The middle row shows staining for Myo7a only. The bottom row shows staining for GFP only. Control photomicrographs were taken from untreated (non-AAV infected) explants. [Figure 17]1 is a bar graph quantifying the percentage of hair cells that were GFP positive in neonatal mouse cochlear explants infected with AAV vectors expressing hypersensitive GFP under the control of various STRC promoters. [Figure 18] 1 is a bar graph quantifying separately the percentage of inner and outer hair cells that were GFP positive in neonatal mouse cochlear explants infected with AAV vectors expressing hypersensitive GFP under the control of various STRC promoters. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] Described herein are compositions and methods for directing transgene expression specifically in hair cells (e.g., cochlear and vestibular hair cells). The invention features STRC promoters capable of directing expression of an expression product (e.g., a protein encoded by a transgene or an RNA molecule, such as an inhibitory RNA molecule) in hair cells that endogenously express STRC (e.g., cochlear hair cells, such as outer hair cells and inner hair cells, and vestibular hair cells, such as type I and type II vestibular hair cells). The invention also features nucleic acid vectors containing such promoters operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein or an inhibitory RNA). The compositions and methods described herein can be used to specifically express a desired expression product (e.g., a protein, an inhibitory RNA, a microRNA, or a component of a gene editing system) in hair cells, and thus the compositions described herein can be administered to a subject (such as a mammalian subject, e.g., a human) to treat disorders caused by dysfunction of cochlear or vestibular hair cells, such as hearing loss and vestibular dysfunction.

[0123] hair cells Hair cells are sensory cells of the auditory and vestibular systems present in the inner ear. Cochlear hair cells are sensory cells of the auditory system and are composed of two main cell types: inner hair cells, which are involved in sound detection, and outer hair cells, which are thought to amplify soft sounds. Vestibular hair cells are located in the end organs of the semicircular canals and otolithic organs of the inner ear and are involved in the sense of movement that contributes to balance and spatial orientation. Hair cells are so named for their stereocilia, which project from the apical surface of the cell to form a hair cell bundle. Tilting of the stereocilia (e.g., by sound waves in cochlear hair cells or by rotational or linear acceleration in vestibular hair cells) causes mechano-gated ion channels to open, which allows the hair cells to release neurotransmitters to activate nerves, thereby transducing mechanical sound or movement signals into electrical signals that can be transmitted to the brain. Cochlear hair cells are essential for normal hearing, and damage or loss of cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are implicated in hearing loss and hearing loss. Damage or loss of vestibular hair cells and genetic mutations that disrupt vestibular hair cell function are implicated in vestibular dysfunction, such as dizziness, vertigo, loss of balance, bilateral vestibular disorders (also known as bilateral vestibular hypofunction), oscillopsia, and balance disorders. Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss and vestibular dysfunction, but the field is in need of methods to specifically target the nucleic acid vectors used in gene therapy to hair cells.

[0124] The present invention is based, in part, on the discovery of STRC promoter sequences that can be used to drive gene expression in hair cells that endogenously express STRC (e.g., cochlear and vestibular hair cells). These STRC promoters are hair cell specific promoters. Thus, the compositions and methods described herein can be used to cause hair cells to express a desired expression product, such as a gene involved in hair cell development, hair cell function, hair cell fate determination, hair cell regeneration, hair cell survival, or hair cell maintenance, or a gene known to be disrupted, e.g., mutated, in a subject with hearing loss or vestibular dysfunction, to treat a subject with or at risk of developing hearing loss (e.g., sensorineural hearing loss), tinnitus, or vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopia, or balance disorders).

[0125] Stereocilin Stereocilin (also known as DFNB16) is a protein encoded by the STRC gene on chromosome 15q15, which contains 29 exons spanning approximately 19 kb of the genome. The STRC gene is tandemly duplicated, where the second copy contains a premature stop codon in exon 20, thereby giving rise to a STRC pseudogene. Previous studies have identified two frameshift mutations and multiple deletions within the full-length copy of STRC in two families with autosomal recessive nonsyndromic sensorineural hearing loss (Verpy et al., Nat. Genet. 29:345-9 (2001)). Stereocilin protein expression is restricted to the stereocilia of hair cell bundles, and stereocilin is thought to form the apical lateral connectors and tectorial adhesion crown required for the normal functioning of the hearing apparatus (Avan et al., PNAS 116:25948-57 (2019); Verpy et al., J. Comp. Neurol. 519:194-210 (2011)). Stereocilin-deficient mice have been shown to exhibit abnormal hair cell bundles with adhesion defects and hearing impairment (Verpy et al., Nature 456:255-8 (2008)).

[0126] The present invention is based, in part, on the discovery of a 500 base pair (bp) region located upstream of the human STRC translation start site and a 537 bp region located upstream of the mouse STRC translation start site that can be used to induce gene expression in hair cells that endogenously express STRC. Thus, the compositions and methods described herein can be used to induce hair cells (e.g., cochlear hair cells, such as outer or inner hair cells, or vestibular hair cells, such as type I or type II hair cells) to express a desired expression product, such as a gene involved in hair cell development, function, cell fate determination, regeneration, survival, or maintenance, or a gene known to be disrupted, e.g., mutated, in subjects with hearing loss or vestibular dysfunction, to treat subjects with or at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopia, or balance disorders). The discovery of the smallest possible STRC promoter that retains endogenous promoter activity would be advantageous for use in nucleic acid vectors, particularly in vectors with limited packaging capacity, such as AAV vectors, which have a maximum packaging capacity believed to be approximately 4.7 kb.

[0127] The compositions and methods described herein include the STRC promoters set forth in Table 2 (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:48) and portions thereof that are capable of expressing a desired expression product (e.g., a transgene) specifically in hair cells that endogenously express STRC (e.g., cochlear and vestibular hair cells), such as polynucleotide sequences having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:48, or to a functional portion of SEQ ID NO:2 or SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 252-537 or 35-530 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 is a portion of a larger SEQ ID NO:2 that includes nucleotides 252-537 of SEQ ID NO:2, e.g., a portion that includes nucleotides 120-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280-560 of SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:48 is a portion of a larger SEQ ID NO:48 that includes nucleotides 280-560 of SEQ ID NO:48, e.g., a portion that includes nucleotides 280-564 of SEQ ID NO:48, nucleotides 124-564 of SEQ ID NO:48, or nucleotides 1-560 of SEQ ID NO:48.In some embodiments, a STRC promoter for use in the compositions and methods described herein includes a portion having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, nucleotides 252-537 of SEQ ID NO:2, nucleotides 120-537 of SEQ ID NO:2, nucleotides 35-530 of SEQ ID NO:2, nucleotides 280-560 of SEQ ID NO:48, nucleotides 280-564 of SEQ ID NO:48, nucleotides 124-564 of SEQ ID NO:48, or nucleotides 1-560 of SEQ ID NO:48. In some embodiments, a STRC promoter for use in the compositions and methods described herein has the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, nucleotides 252-537 of SEQ ID NO:2, nucleotides 120-537 of SEQ ID NO:2, nucleotides 35-530 of SEQ ID NO:2, nucleotides 280-560 of SEQ ID NO:48, nucleotides 280-564 of SEQ ID NO:48, nucleotides 124-560 of SEQ ID NO:48, nucleotides 124-564 of SEQ ID NO:48, or nucleotides 1-560 of SEQ ID NO:48.

[0128] Exemplary STRC promoter sequences are listed in Table 2.

[0129] [Table 2-1]

[0130] [Table 2-2]

[0131] The promoter sequences described above can be included in a nucleic acid vector and operably linked to a polynucleotide encoding a desired expression product (e.g., a polynucleotide encoding a protein of interest or an inhibitory RNA) to direct expression of the expression product specifically in hair cells (e.g., in hair cells that endogenously express STRC, such as cochlear hair cells (e.g., outer and inner hair cells) and vestibular hair cells (e.g., type I and type II vestibular hair cells). In some embodiments, the polynucleotide operably linked to the STRC promoter is a transgene that encodes a protein involved in hair cell function, hair cell development, hair cell fate determination, hair cell regeneration, hair cell survival, or hair cell maintenance, or is a transgene that corresponds to a wild-type version of a gene known to be mutated in subjects with hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopia, or balance disorders). According to the methods described herein, a subject can be administered a composition containing one or more of the aforementioned polynucleotides operably linked to a polynucleotide encoding a desired expression product, e.g., a transgene encoding a protein of interest (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to a STRC promoter, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48). In some embodiments, the protein encoded by the transgene is Actin Gamma 1 (ACTG1), Retinal Fascin Actin-Bundling Protein 2 (FAS-B), or a retinal Fascin Actin-Bundling Protein 2 (FRP ...2, Retinal (FSCN2), Radixin (RDX), POU class 4 homeobox 3 (POU4F3), TRIO and F-actin binding protein (TRIOBP), Taperin (TPRN), Xin actin binding repeat containing 2 (XIRP2), Atonal BHLH transcription factor 1 (ATOH1), transcriptional repressor Growth Factor Independent 1 (GFI1), Cholinergic nicotinic receptor alpha 9 subunit (CHRNA9), Cholinergic nicotinic receptor alpha 10 subunit (CHRNA10), Calcium and integrin binding family member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), MKRN2 Opposite Strand (MKRN2OS), LIM homeobox protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain Containing Cysteine-Rich Protein 1 (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+ Transport 2 (ATPase Plasma Membrane Ca2+ Transporting2) (ATP2B2), calcium and integrin binding family member 2 (CIB2), voltage-dependent calcium channel auxiliary subunit alpha 2 delta 4 (CACNA2D4), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), peripherin 2 (PRPH2), solute carrier family 8 member A2 (SLC8A2), zinc finger CCHC-type containing protein 12 (ZCCHC12), leucine rich transmembrane and O-methyltransferase domain containing Containing (LRTOMT2, LRTOMT1), USH1 protein network component harmonin (USH1C), solute carrier family 26 member 5 (SLC26A5), Piezo-type mechanosensitive ion channel component 2 (PIEZO2), extracellular leucine rich repeat and fibronectin type III domain containing 1 (ELFN1), tetratricopeptide repeat protein 24 (TTC24), dystrotelin (DYTN), coiled-coil glutamic acid rich protein 2 (CCER2), leucine rich repeat and transmembrane domain containing protein 2 (LRTM2), voltage-gated potassium channel subfamily A member 10 (KCNA10), clarin 1 (CLRN1), clarin 2 (CLRN2), SKI family transcriptional corepressor 1 (SKI Family Transcriptional Core ... Corepressor 1) (SKOR1), Tctex1 domain-containing protein 1 (TCTEX1D1), Fc receptor-like B (FCRLB), glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2), serpin family E member 3 (SERPINE3), Nescient helix-loop-helix 1 (NescientHelix-loop helix 1 (NHLH1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), activating transcription factor 6 (ATF6), eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), serine / threonine protein kinase / endoribonuclease IRE1 (IRE1), Whirlin (WHRN), oncomodulin (OCM), LIM homeobox 1 (Isl1), transmembrane and tetratricopeptide repeat-containing 4 (TMTC4), or binding immunoglobulin protein (BIP), or voltage-gated potassium channel subfamily Q member 4 (KCNQ4).

[0132] In some embodiments, a STRC promoter described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) is operably linked to a polynucleotide encoding the N-terminal portion of stereocillin (e.g., a polynucleotide encoding the N-terminal portion of SEQ ID NO:3 or SEQ ID NO:4) and incorporated into a first vector in a two-vector system. The full-length stereocillin coding sequence is too large to be included in the types of vectors (e.g., AAV vectors) commonly used for gene therapy, but this problem can be solved by splitting the stereocillin coding sequence between two different nucleic acid vectors, allowing the full-length stereocillin sequence to be reconstituted in cells (e.g., hair cells). Such a two-vector system can be used to treat sensorineural hearing loss or vestibular dysfunction in a subject by administering to the inner ear of the subject a first nucleic acid vector containing a polynucleotide encoding the N-terminal portion of the stereocillin protein and a second nucleic acid vector containing a polynucleotide encoding the C-terminal portion of the stereocillin protein. These two-vector systems can be used to treat subjects with one or more mutations in the STRC gene, such as mutations in STRC that reduce stereocillin expression, reduce stereocillin function, or are associated with hearing loss or vestibular dysfunction. When the first and second nucleic acid vectors are administered in a composition, the polynucleotide encoding the N-terminal portion and the polynucleotide encoding the C-terminal portion of stereocillin can combine within a cell (e.g., a human cell, e.g., a cochlear or vestibular hair cell) to form a single polynucleotide containing the full-length stereocillin coding sequence (e.g., via homologous recombination and / or splicing).

[0133] The nucleic acid vectors of the two-vector system described herein include polynucleotide sequences encoding WT stereocillin, or variants thereof, such as polynucleotide sequences encoding proteins that, when combined, have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of WT mammalian (e.g., human or mouse) stereocillin. The polynucleotides used in the two-vector system described herein encode the N-terminal and C-terminal portions of the amino acid sequence of stereocillin in Table 3 below (e.g., two portions that, when combined, encode the full-length stereocillin amino acid sequence in Table 3, e.g., SEQ ID NO:3 or SEQ ID NO:4).

[0134] [Table 3-1]

[0135] [Table 3-2]

[0136] [Table 3-3]

[0137] [Table 3-4]

[0138] [Table 3-5]

[0139] [Table 3-6]

[0140] [Table 3-7]

[0141] [Table 3-8]

[0142] According to the methods described herein, a composition containing a first nucleic acid vector and a second nucleic acid vector containing, respectively, an N-terminal portion and a C-terminal portion of a polynucleotide sequence encoding an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conservative amino acid substitutions) relative to SEQ ID NO:3 or SEQ ID NO:4, provided that the encoded stereocillin analog retains the therapeutic function of WT stereocillin. In some embodiments, up to 10% of the amino acids in the N-terminal portion of the stereocillin protein and up to 10% of the amino acids in the C-terminal portion of the stereocillin protein may be replaced by conservative amino acid substitutions. The stereocillin protein may be encoded by a polynucleotide having the sequence of SEQ ID NO:5 or SEQ ID NO:6. The stereocillin protein may also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) that is known to be non-pathogenic in human subjects. The stereocillin protein may be a human stereocillin protein or a homolog of the human stereocillin protein from another mammalian species (e.g., mouse, rat, cow, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig, or other mammal).

[0143] In some embodiments, the first and second nucleic acid vectors are the first and second vectors in a dual vector expression system (e.g., an overlapping dual vector, a trans-splicing dual vector, or a dual hybrid vector). In some embodiments, the first and second nucleic acid vectors are the first and second vectors in an intein expression system. In some embodiments, the first and second vectors are the first and second vectors in a ribozyme expression system (e.g., a system in which the first nucleic acid vector contains a polynucleotide encoding an N-terminal portion of a stereocillin and a 3' ribozyme, and the second vector contains a polynucleotide encoding a C-terminal portion of a stereocillin and a 5' ribozyme). In the ribozyme expression system, the 3' ribozyme and the 5' ribozyme can catalyze themselves from the first and second RNA molecules produced during transcription to produce 3' and 5' ends, which can be linked to form an RNA molecule containing the coding region of the first RNA molecule and the coding region of the second RNA molecule. In some embodiments, the 3' ribozyme is a member of the HDV (hepatitis delta virus) family of ribozymes, and the 5' ribozyme is a member of the HH (hammerhead) family of ribozymes. Exemplary ribozyme expression systems are described in International Publication No. WO2021158964A1, which is incorporated herein by reference.

[0144] Dual vector expression system Overlapping double vector One approach for expressing large proteins in mammalian cells (e.g., hair cells) involves the use of overlapping double vectors. This approach is based on the use of two nucleic acid vectors, each of which contains a portion of the polynucleotide that encodes the protein of interest and has a defined sequence overlap region with the other portion of the polynucleotide. Homologous recombination can occur at the overlap region, resulting in the formation of a single polynucleotide that encodes the full-length protein of interest (e.g., the stereocillin protein of SEQ ID NO:3 or SEQ ID NO:4).

[0145] An overlapping double vector for use in the methods and compositions described herein contains at least 200 bases (b) of overlapping sequence (e.g., at least 200b, 300b, 400b, 500b, 600b, 700b, 800b, 900b, 1.0 kilobase (kb), 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb or more of overlapping sequence). The nucleic acid vector is designed such that the overlapping region is at or near a central position within the stereocillin-encoding polynucleotide that corresponds to approximately half the length of the stereocillin-encoding polynucleotide, with equal amounts of overlap on either side of the central position. The center of the overlapping region can also be selected based on the size of the promoter and the location of the sequence element of interest in the stereocillin-encoding polynucleotide. In some embodiments, the polynucleotide encoding stereocillin is split into two approximately equal lengths with some overlap (e.g., 200b, 250b, 300b, 350b, 400b, 450b, 500b, 600b, 700b, 800b, 900b, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, or more), with the 5' half of the polynucleotide encoding the N-terminal portion of the stereocillin protein and the 3' half of the polynucleotide encoding the C-terminal portion of the stereocillin protein. Nucleic acid vectors for use in the methods and compositions described herein are also designed such that approximately half of the polynucleotides encoding stereocillin are contained within each vector (e.g., each vector contains polynucleotides encoding approximately half of the stereocillin protein).

[0146] In some embodiments, the first nucleic acid vector encodes the N-terminal portion of the stereocillin protein. In some embodiments, the second nucleic acid vector encodes the C-terminal portion of the stereocillin protein. In some embodiments, the stereocillin protein has a sequence of SEQ ID NO:3 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the stereocillin protein has a sequence of SEQ ID NO:4 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the polynucleotide encoding the full-length human stereocillin protein has a sequence of SEQ ID NO:5 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the polynucleotide having at least 85% sequence identity to SEQ ID NO:5 encodes the stereocillin protein of SEQ ID NO:3. In some embodiments, the polynucleotide encoding the full-length mouse stereocillin protein has a sequence of SEQ ID NO:6 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, a polynucleotide having at least 85% sequence identity to SEQ ID NO:6 encodes the stereocillin protein of SEQ ID NO:4.

[0147] One exemplary overlapping dual vector system includes a first nucleic acid vector comprising a STRC promoter as described herein above (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of SEQ ID NO:3 or SEQ ID NO:4) that includes 500 bp immediately 3' to a position selected as the central position, the STRC promoter being at least 50 bp longer than the first nucleic acid vector, and at least 50 bp longer than the first nucleic acid vector ... The nucleic acid vectors include a first nucleic acid vector containing a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and a second nucleic acid vector containing a C-terminal portion of a polynucleotide encoding a stereocillin protein, the second nucleic acid vector including 500 bp adjacent 5' to the position selected as the central position, and a poly(A) sequence (e.g., bovine growth hormone (bGH) poly(A) signal sequence). The nucleic acid vector can optionally contain a STRC untranslated region (UTR) that is not part of the STRC promoter disclosed herein. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 1 or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 1. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 2 or a functional portion thereof or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 2 or a functional portion thereof.In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:2 that is capable of controlling expression of the STRC gene. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 252-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 120-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 35-530 of SEQ ID NO:2. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO:48 or a functional portion thereof, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:48 or a functional portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:48 that is capable of controlling expression of the STRC gene. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280 to 560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280 to 564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124 to 560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124 to 564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 1 to 560 of SEQ ID NO:48.

[0148] Trans-splicing dual vector A second approach for expressing large proteins in mammalian cells involves the use of trans-splicing dual vectors. In this approach, two nucleic acid vectors are used that contain separate nucleic acid sequences, with the polynucleotide encoding the N-terminal portion of the protein of interest not overlapping with the polynucleotide encoding the C-terminal portion of the protein of interest. Instead, the first nucleic acid vector contains a splice donor sequence 3' to the polynucleotide encoding the N-terminal portion of the protein of interest, and the second nucleic acid vector contains a splice acceptor sequence 5' to the polynucleotide encoding the C-terminal portion of the protein of interest. When the first and second nucleic acids are present in the same cell, their ITRs can be linked to form a single nucleic acid structure in which the linked ITRs are located between the splice donor and splice acceptor. Trans-splicing then occurs during transcription to generate a nucleic acid molecule in which the polynucleotide encoding the N-terminal portion of the protein of interest and the polynucleotide encoding the C-terminal portion of the protein of interest are contiguous, thereby forming a full-length coding sequence.

[0149] The trans-splicing dual vectors for use in the methods and compositions described herein are designed such that approximately half of the stereocillin coding sequence is contained within each vector (e.g., each vector contains a polynucleotide encoding approximately half of the stereocillin protein, as described above). The determination of how to split the polynucleotide sequence between the two nucleic acid vectors is based on the size of the promoter and the location of the sequence element of interest (e.g., an exon of the STRC gene) in the polynucleotide encoding the stereocillin protein. The first vector in the trans-splicing dual vector system can contain a promoter sequence (e.g., a STRC promoter sequence, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, or a functional portion of SEQ ID NO:2 or SEQ ID NO:48) 5' of the polynucleotide encoding the N-terminal portion of the stereocillin protein (e.g., the N-terminal portion of the stereocillin protein of SEQ ID NO:3 or SEQ ID NO:4). The nucleic acid vector can optionally contain a STRC UTR that is not part of the promoter described herein (e.g., one or both of the 5' and 3' STRC UTRs, e.g., a full-length UTR).One exemplary trans-splicing dual vector system for use in the compositions and methods described herein includes a first nucleic acid vector, comprising a STRC promoter as described herein above (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of a human stereocillin protein, e.g., SEQ ID NO:3), The present invention also includes a first nucleic acid vector containing a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and a splice donor sequence 3' of a polynucleotide sequence, and a second nucleic acid vector containing a splice acceptor sequence and a poly(A) sequence 5' of a polynucleotide encoding a C-terminal portion of a stereocillin protein (e.g., the C-terminal portion of a human stereocillin protein, e.g., SEQ ID NO: 3).The alternative trans-splicing dual vector system includes a first nucleic acid vector, comprising a STRC promoter as described herein above (e.g., a polynucleotide having at least 85% sequence identity (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of a mouse stereocillin protein, e.g., SEQ ID NO:4). The nucleic acid vectors include a first nucleic acid vector containing a STRC promoter having a sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and a splice donor sequence 3' of a polynucleotide sequence, and a second nucleic acid vector containing a splice acceptor sequence and a poly(A) sequence 5' of a polynucleotide encoding a C-terminal portion of a stereocillin protein (e.g., the C-terminal portion of a mouse stereocillin protein, e.g., SEQ ID NO:4). In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 1 or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 1. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 2 or a functional portion thereof or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 2 or a functional portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO: 2 that can control expression of the STRC gene.In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 252-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 120-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 35-530 of SEQ ID NO:2. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO:48 or a functional portion thereof, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:48 or a functional portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:48 that is capable of controlling expression of the STRC gene. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280 to 560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280 to 564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124 to 560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124 to 564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 1 to 560 of SEQ ID NO:48.

[0150] These nucleic acid vectors may also contain a full length 5' STRC UTR and / or a full length 3' STRC UTR in the first and second nucleic acid vectors, respectively, that is not part of the promoter described herein (e.g., the first nucleic acid vector may contain the 5' human STRC UTR in a dual vector system encoding human stereocillin and the 5' mouse UTR in a dual vector system encoding mouse stereocillin, and the second nucleic acid vector may contain the 3' human STRC UTR in a dual vector system encoding human stereocillin and the 3' mouse STRC UTR in a dual vector system encoding mouse stereocillin). To accommodate the STRC UTR, the stereocillin coding sequence can be split at a different position than that used to split the stereocillin coding sequence in a trans-splicing dual vector system that does not include a STRC UTR (e.g., the stereocillin coding sequence can be split at a position such that the first vector can accommodate the length of the 5'UTR, the length of the promoter sequence, and the length of the sequence encoding the N-terminal portion of stereocillin, and / or the second vector can accommodate the length of the C-terminal portion of stereocillin and the length of the 3'UTR).

[0151] In some embodiments, the stereocillin protein has a sequence of SEQ ID NO: 3 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the stereocillin protein has a sequence of SEQ ID NO: 4 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the polynucleotide encoding the full-length human stereocillin protein has a sequence of SEQ ID NO:5 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the polynucleotide having at least 85% sequence identity to SEQ ID NO:5 encodes the stereocillin protein of SEQ ID NO:3. In some embodiments, the polynucleotide encoding the full-length mouse stereocillin protein has a sequence of SEQ ID NO:6 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6. In some embodiments, a polynucleotide having at least 85% sequence identity to SEQ ID NO:6 encodes the stereocillin protein of SEQ ID NO:4.

[0152] Double hybrid vectors A third approach for expressing large proteins in mammalian cells (e.g., hair cells) involves the use of a double hybrid vector. This approach combines elements of the overlapping double vector strategy with elements of the trans-splicing strategy, and features both an overlapping region capable of homologous recombination occurring, as well as splice donor and splice acceptor sequences. In a double hybrid vector system, the overlapping region is not part of the polynucleotide sequence encoding the protein of interest, but is a recombinogenic region contained in both the first and second nucleic acid vectors, where the polynucleotide encoding the N-terminal portion of the protein of interest does not overlap with the polynucleotide encoding the C-terminal portion of the protein of interest. The recombinogenic region is 3' of the splice donor sequence in the first nucleic acid vector and 5' of the splice acceptor sequence in the second nucleic acid sequence. The first and second nucleic acid sequences can then be ligated to form a single sequence based on one of two mechanisms: (1) recombination at the overlapping region, or (2) concatemerization of the ITRs. The remaining recombinogenic region(s) and / or concatemerized ITRs may be removed by splicing, resulting in the formation of a contiguous polynucleotide sequence encoding the full-length protein of interest. Recombinogenic regions, splice donor sequences, and splice acceptor sequences that may be used in the compositions and methods described herein include those known to those of skill in the art. Exemplary recombinogenic regions include fragments of the F1 phage AK gene and alkaline phosphatase (AP) gene, as described in U.S. Pat. Nos. 10,494,645 and 8,236,557, which are incorporated herein by reference. In some embodiments, the AP gene fragment is It has the sequence of CCCC GGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCCGTATAGGAGGACCGTGTAGGCCTTCCTGTCCCGGGCCTTGCCAGCGGCCAGCCCGATGAAGGAGCTCCCTCGCAGGGGGTAGCCTCCGAAGGAGAAGACGTGGGAGTGGTCGGCAGTGACGAGGCTCAGCGTGTCCTCCTCGCTGGTGAGCTGGCCCGCCCTCTCAATGGCGTCGTCGAACATGATCGTCTCAGTCAGTGCCCGGTAAGCCCTGCTTTCATGATGACCATGGTCGATGCGACCACCCTCCACGAAGAGGAAGAAGCCGCGGGGGTGTCTGCTCAGCAGGCGCAGGGCAGCCTCTGTCATCTCCATCAGGGAGGGGTCCAGTGTGGAGTCTCGGTGGATCTCGTATTTCATGTCTCCAGGCTCAAAGAGACCCATGAGATGGGTCACAGACGGGTCCAGGGAAGCCTGCATGAGCTCAGTGCGGTTCCACACGTACCGGGCACCCTGGCGTTCGCCGAGCCATTCCTGCACCAGATTCTTCCCGTCCAGCCTGGTCCCACCTTGGCTGTAGTCATCTGGGTACTCAGGGTCTGGGGTTCCCATGCGAAACATGTACTTTCGGCCTCCA (SEQ ID NO: 42).

[0153] In some embodiments, the AP gene fragment is CCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGCCGGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCG It has a sequence of CTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCCGTATAGGAGGACCGTGTAGGCCTTCCTGTCCCGGGCCTTGCCAGCGGCCAGCCCGATGAAGGAGCTCCCTCGCAGGGGGTAGCCTCCGAAGGAGAAGACGTGGGAGTGGTCGGCAGTGACGAGGCTCAGCGTGTCCTCCTCGCTGGTGA (SEQ ID NO: 43).

[0154] In some embodiments, the AP gene fragment comprises: GCTGGCCCGCCCTCTCAATGGCGTCGTCGAACATGATCGTCTCAGTCAGTGCCCGGTAAGCCCTGCTTTCATGATGACCATGGTCGATGCGACCACCCTCCACGAAGAGGAAGAAGCCGCGGGGGTGTCTGCTCAGCAGGCGCAGGGCAGCCTCTGTCATCTCCATCAGGGAGGGGTCCAGTGTGGAGTCTCGGTGGATCTCGTATTTCATGTCTCCAGGCT CAAAGAGACCCATGAGATGGGTCACAGACGGGTCCAGGGAAGCCTGCATGAGCTCAGTGCGGTTCCACACGTACCGGGCACCCTGGCGTTCGCCGAGCCATTCCTGCACCAGATTCTTCCCGTCCAGCCTGGTCCCACCTTGGCTGTAGTCATCTGGGTACTCAGGGTCTGGGGTTCCCATGCGAAACATGTACTTTCGGCCTCCA (SEQ ID NO: 44).

[0155] In some embodiments, the AP gene fragment comprises: CCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGCCGCGCGCGAAC It has a sequence of ACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTC (SEQ ID NO: 45).

[0156] In some embodiments, the AP gene fragment comprises: CGTATAGGAGGACCGTGTAGGCCTTCCTGTCCCGGGCCTTGCCAGCGGCCAGCCCGATGAAGGAGCTCCCTCGCAGGGGGTAGCCTCCGAAGGAGAAGACGTGGGAGTGGTCGGCAGTGACGAGGCTCAGCGTGTCCTCCTCGCTGGTGAGCT It has a sequence of GGCCCGCCCTCTCAATGGCGTCGTCGAACATGATCGTCTCAGTCAGTGCCCGGTAAGCCCTGCTTTCATGATGACCATGGTCGATGCGACCACCCTCCACGAAGAGGAAGAAGCCGCGGGGGTGTCTGCTCAGCAGG (SEQ ID NO: 46).

[0157] In some embodiments, the AP gene fragment comprises: It has the sequence CGCAGGGCAGCCTCTGTCATCTCCATCAGGGAGGGGTCCAGTGTGGAGTCTCGGTGGATCTCGTATTTCATGTCTCCAGGCTCAAAGAGACCATGAGATGGGTCACAGAGGGTCCAGGGAAGCCTGCATGAGCTCAGTGCGGTTCCACACGTACCGGCACCCTGGCGTTCGCCGAGCCATTCCTGCCACCAGATTCTTCCCGTCCCAGCTGGTCCCACCTTGGCTGTAGTCATCTGGGTACTCAGGGTCTGGGGTTCCCATGCGAAACATGTACTTTCGCCCCTCCA (sequence number 47).

[0158] The double hybrid vectors for use in the methods and compositions described herein are designed such that approximately half of the stereocillin coding sequence is contained within each vector (e.g., each vector contains a polynucleotide encoding approximately half of the stereocillin protein). The decision of how to split the polynucleotide sequence between the two nucleic acid vectors is based on the size of the promoter and the location of the sequence element of interest (e.g., an exon of the STRC gene) in the polynucleotide encoding the stereocillin protein. The first vector in the double hybrid vector system can contain a promoter sequence 5' to the polynucleotide encoding the N-terminal portion of the stereocillin protein. The nucleic acid vector can optionally contain a STRC UTR (e.g., full length 5'UTR and / or full length 3'UTR) that is not part of the promoter described herein.One exemplary two-hybrid vector system includes a first nucleic acid vector, comprising a STRC promoter as described herein above (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48), operably linked to an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of human stereocillin, e.g., SEQ ID NO:3), and a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 15 The present invention also includes a first nucleic acid vector comprising a STRC promoter having a sequence identity (e.g., 96%, 97%, 98%, 99% or more) with a polynucleotide sequence of the STRC promoter, a splice donor sequence 3' to the polynucleotide sequence, and a recombinogenic region 3' to the splice donor sequence; and a second nucleic acid vector comprising a recombinogenic region (e.g., the same as the recombinogenic region contained in the first vector), a splice acceptor sequence 3' to the recombinogenic region, a polynucleotide encoding a C-terminal portion of a stereocillin protein (e.g., human stereocillin, e.g., the C-terminal portion of SEQ ID NO: 3) 3' to the splice acceptor sequence, and a poly(A) sequence. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 1, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 1. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO: 2 or a functional portion thereof, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 2 or a functional portion thereof.In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:2 that is capable of controlling expression of the STRC gene. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 252-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 120-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 35-530 of SEQ ID NO:2. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO:48 or a functional portion thereof, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:48 or a functional portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:48 that is capable of controlling expression of the STRC gene. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280-560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280-564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124-560 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124-564 of SEQ ID NO:48. In some embodiments, a functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 1-560 of SEQ ID NO:48. The first and second nucleic acid vectors can each also contain a full length 5' STRC UTR and / or a full length 3' STRC UTR (e.g., a human STRC 5'UTR can be included in the first nucleic acid vector and a human STRC 3'UTR can be included in the second nucleic acid vector).

[0159] Another exemplary two-hybrid vector system that includes a STRC promoter includes a first nucleic acid vector, the first nucleic acid vector comprising a STRC promoter as described herein above (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of mouse stereocillin, e.g., SEQ ID NO:4). , 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), a splice donor sequence 3' of the polynucleotide sequence, and a recombinogenic region 3' of the splice donor sequence, and a second nucleic acid vector, the second nucleic acid vector comprising a recombinogenic region (e.g., the same as the recombinogenic region contained in the first vector), a splice acceptor sequence 3' of the recombinogenic region, a polynucleotide encoding a C-terminal portion of a stereocillin protein 3' of the splice acceptor sequence (e.g., mouse stereocillin, e.g., the C-terminal portion of SEQ ID NO: 4), and a poly(A) sequence. The first and second nucleic acid vectors can each also contain a full-length 5' STRC UTR and / or a full-length 3' STRC UTR (e.g., a mouse STRC 5' UTR can be included in the first nucleic acid vector and a mouse STRC 3' UTR can be included in the second nucleic acid vector). To accommodate the STRC UTR, the stereocillin coding sequence can be split at a different location than in a two-hybrid vector system that does not contain a STRC UTR.

[0160] In some embodiments, the stereocillin protein has a sequence of SEQ ID NO: 3 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the stereocillin protein has a sequence of SEQ ID NO: 4 or has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto. In some embodiments, the polynucleotide encoding the full-length human stereocillin protein has a sequence of SEQ ID NO:5 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the polynucleotide having at least 85% sequence identity to SEQ ID NO:5 encodes the stereocillin protein of SEQ ID NO:3. In some embodiments, the polynucleotide encoding the full-length mouse stereocillin protein has a sequence of SEQ ID NO:6 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6. In some embodiments, a polynucleotide having at least 85% sequence identity to SEQ ID NO:6 encodes the stereocillin protein of SEQ ID NO:4.

[0161] In some embodiments, a first vector in a two-hybrid vector system for use in the compositions and methods described herein contains nucleotides 1-1800 of a polynucleotide encoding mouse stereocillin (e.g., nucleotides 1-1800 of SEQ ID NO: 6) and a second vector contains the remaining nucleotides of a polynucleotide encoding mouse stereocillin (e.g., nucleotides 1801-5430 of SEQ ID NO: 6). In some embodiments, the polynucleotide encoding mouse stereocillin is separated between the first and second vectors at an exon boundary of STRC. In some embodiments, the exon boundary is the exon 4 / exon 5 boundary, the exon 5 / exon 6 boundary, the exon 6 / exon 7 boundary, or the exon 7 / exon 8 boundary. In a double hybrid vector system in which the mouse STRC coding sequence is split at the exon 4 / exon 5 boundary, the first vector contains nucleotides 1-2247 of the polynucleotide encoding stereocillin (e.g., nucleotides 1-2247 of SEQ ID NO: 6) and the second vector contains the remaining nucleotides of the polynucleotide encoding stereocillin (e.g., nucleotides 2248-5430 of SEQ ID NO: 6). In a double hybrid vector system in which the mouse STRC coding sequence is split at the exon 5 / exon 6 boundary, the first vector contains nucleotides 1-2310 of the polynucleotide encoding stereocillin (e.g., nucleotides 1-2310 of SEQ ID NO: 6) and the second vector contains the remaining nucleotides of the polynucleotide encoding stereocillin (e.g., nucleotides 2311-5430 of SEQ ID NO: 6). In a double hybrid vector system in which the mouse STRC coding sequence is split at the exon 6 / exon 7 boundary, a first vector contains nucleotides 1 to 2421 of the polynucleotide encoding stereocillin (e.g., nucleotides 1 to 2421 of SEQ ID NO:6) and a second vector contains the remaining nucleotides of the polynucleotide encoding stereocillin (e.g., nucleotides 2422 to 5430 of SEQ ID NO:6).In a double hybrid vector system in which the mouse STRC coding sequence is split at the exon 7 / exon 8 boundary, a first vector contains nucleotides 1 to 2588 of the polynucleotide encoding stereocillin (e.g., nucleotides 1 to 2588 of SEQ ID NO:6) and a second vector contains the remaining nucleotides of the polynucleotide encoding stereocillin (e.g., nucleotides 2589 to 5430 of SEQ ID NO:6).

[0162] The double hybrid vector used in the methods and compositions described herein can optionally include a degradation signal sequence in both the first and second nucleic acid vectors. The degradation signal sequence can be included to prevent or reduce the expression of a portion of the stereocillin protein from polynucleotides that could not be recombined and / or spliced. The degradation signal sequence is located 3' of the recombination-inducing region in the first nucleic acid vector, and is located between the recombination-inducing region and the splice acceptor in the second nucleic acid vector. Suitable degradation signal sequences that can be used in the compositions and methods described herein are known in the art and are described, for example, in International Application Publication No. WO2016 / 139321, which is incorporated herein by reference.

[0163] In some embodiments, the first member of the dual vector system includes a STRC promoter (operably linked to a polynucleotide encoding the N-terminal portion of a stereocillin protein) of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 including nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 including nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48. The polynucleotide sequence encoding the N-terminal portion of the stereocillin protein can be partially or completely codon-optimized for expression. In some embodiments, the first member of the dual vector system includes a splice donor sequence. In some embodiments, the first member of the dual vector system includes an AP gene fragment described herein (e.g., any one of SEQ ID NOs: 42-47, e.g., SEQ ID NO: 45). In some embodiments, the first member of the dual vector system is flanked on both the 5' and 3' sides by ITRs. In some embodiments, the flanking ITRs are any variant of the AAV2 terminal inverted repeat sequence that can be encapsidated by a plasmid carrying the AAV2 Rep gene. It will be understood by those skilled in the art that for any given pair of ITR sequences in the transfer plasmid used to generate the viral vector (typically by transfecting the transfer plasmid into cells with other plasmids carrying the necessary AAV genes for viral vector formation), the corresponding sequences in the viral vector may be altered so that the ITRs adopt a "flip" or "flop" orientation upon recombination.Thus, the sequences of the ITRs in a transfer plasmid are not necessarily the same sequences as those found in the viral vector prepared therefrom.

[0164] In some embodiments, the second member of the dual vector system includes a polynucleotide encoding a C-terminal portion of a stereocillin protein followed immediately by a stop codon. The polynucleotide sequence encoding the C-terminal portion of the stereocillin protein can be partially or fully codon optimized for expression. In some embodiments, the second member of the dual vector system includes a splice acceptor sequence. In some embodiments, the second member of the dual vector system includes an AP gene fragment described herein (e.g., any one of SEQ ID NOs: 42-47, e.g., SEQ ID NO: 45). In some embodiments, the second member of the dual vector system includes a poly(A) sequence. In certain embodiments, the second member of the dual vector system is flanked on both the 5' and 3' sides by ITRs. In some embodiments, the flanking ITRs are any variant of the AAV2 ITR that can be encapsidated by a plasmid carrying the AAV2 Rep gene. One of skill in the art will understand that for any given pair of terminal inverted repeat sequences in a transfer plasmid used to generate a viral vector (typically by transfecting the transfer plasmid into cells along with other plasmids carrying the necessary AAV genes for viral vector formation), the corresponding sequences in the viral vector may be altered such that, upon recombination, the ITRs "flip" or "flop" in orientation. Thus, the sequences of the ITRs in the transfer plasmid will not necessarily be the same sequences as those found in the viral vector prepared therefrom.

[0165] To generate a nucleic acid vector (e.g., an AAV vector) for administration, a transfer plasmid (e.g., a plasmid containing a DNA sequence to be delivered by a nucleic acid vector, e.g., a DNA sequence to be delivered by an AAV) can be co-delivered into a producer cell with a helper plasmid (e.g., a plasmid providing proteins necessary for AAV production) and a rep / cap plasmid (e.g., a plasmid providing AAV capsid proteins and proteins that insert the transfer plasmid DNA sequence into the capsid shell). The nucleic acid vectors (e.g., a nucleic acid vector (e.g., an AAV vector) containing a polynucleotide encoding an N-terminal portion of a stereocillin protein and a nucleic acid vector (e.g., an AAV vector) containing a polynucleotide encoding a C-terminal portion of a stereocillin protein) can be combined (e.g., into a single formulation) prior to administration.

[0166] Other exemplary pairs of overlapping, trans-splicing, and double-hybrid vectors are listed in Table 4 below.

[0167] [Table 4-1]

[0168] [Table 4-2]

[0169] [Table 4-3]

[0170] Intein Expression System Another gene therapy approach to express large proteins in mammalian cells involves the use of inteins. Inteins, also known as "protein introns," are portions of proteins that are typically 100-900 amino acid residues long and capable of self-excision and ligation of N- and C-terminal residues of adjacent protein fragments ("exteins"). Inteins can be divided into three different classes, including maxi-inteins, mini-inteins, and split-inteins. Maxi-inteins refer to the N- and C-terminal splicing regions of a protein that are interrupted by a homing endonuclease domain (HEG). HEGs refer to a class of endonucleases that are encoded as standalone genes within introns, as protein fusions with other proteins, or as self-splicing inteins. HEGs generally hydrolyze select DNA regions that are very scarce. When HEGs hydrolyze a DNA fragment, the gene encoding the HEG typically incorporates itself into the cleavage site, thereby increasing its allele frequency. "Mini-intein" refers to an N-terminal splicing domain and a C-terminal splicing domain that lack the HEG domain. "Split intein" refers to an intein that is transcribed and translated as two separate polypeptides that are linked to an extein. Alanine inteins are another class of inteins that have a splice junction at alanine rather than cysteine ​​or serine.

[0171] The splicing domain of inteins contains two subdomains, the N-terminal splicing domain and the C-terminal splicing domain, which contain conserved motifs with conserved residues that mediate splicing activity. The N-terminal splicing domain contains the A, N2, B, and N4 structural motifs, and the C-terminal splicing domain contains the F and G motifs. The A motif contains Cys / Ser or Thr as conserved residues, the B motif contains His and Thr residues, the F motif contains Asp and His residues, and the G motif has two conserved residues including a penultimate His and a terminal Asn. The C, D, E, and H motifs are commonly associated with the HEG domain in maxi-inteins.

[0172] Intein splicing falls within three distinct strategies: 1) class 1 (or classical / canonical) intein splicing, which involves (a) an (NS / NO) acyl shift that converts the peptide bond at the N-terminal splice junction to a thio(ester) bond, (b) a transesterification reaction that forms a branched intermediate, (c) an Asn cyclization that removes the branched intermediate by cleaving the C-terminal splice junction, and (d) a second (SN / ON) acyl shift that links adjacent extein segments via the formation of an amide bond; 2) class 2 inteins (also known as alanine inteins), which skip step (a) of the classical splicing reaction; and 3) a class 3 mechanism, which involves the formation of two branched intermediates.

[0173] Among the various intein systems mentioned above, the split intein trans-splicing approach has been demonstrated to be successful in overcoming the size limitations of conventional gene therapy vectors (e.g., AAV, which has a maximum size limit of about 4.7 kb). For example, Subramanyam et al. (PNAS 110:15461-6 (2013)) used a split intein system to reconstitute the α1C-subunit of the L-type calcium channel in cardiomyocytes from two separate parts. Similarly, Truong et al. (Nucleic Acids Res. 43:6450-8 (2015)) demonstrated successful reconstitution of two separate parts of the Cas9 protein using a split intein system. Thus, the present disclosure provides a split intein trans-splicing system for packaging and delivery of a stereocillin coding sequence operably linked to the STRC promoter. This method allows two separate polynucleotides, each containing approximately one-half of the STRC gene and each containing a polynucleotide sequence encoding an N-intein fragment or a C-intein fragment, to be expressed from two separate expression vectors (e.g., any one of the nucleic acid vectors disclosed herein) and post-translationally reconstituted to generate a full-length stereocillin protein. Such a system can be incorporated into a nucleic acid expression vector disclosed herein, such as, for example, a rAAV vector.

[0174] In one example, the disclosure provides a two-vector split intein system, comprising: a) a first nucleic acid vector containing a polynucleotide that includes a sequence encoding an N-terminal portion of a human stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3), wherein the sequence encoding the N-terminal portion of the human stereocillin protein is provided at its 3' end with an in-frame polynucleotide sequence encoding an N-intein; and b) a second vector, the second vector comprising a polynucleotide comprising a sequence encoding a C-terminal portion of a human stereocillin protein (e.g., a C-terminal portion of SEQ ID NO: 3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 3), wherein the sequence encoding the C-terminal portion of the human stereocillin protein comprises at its 5' end an in-frame polynucleotide sequence encoding a C-intein.

[0175] In another example, the disclosure provides a two-vector split intein system, comprising: a) a first vector containing a polynucleotide that includes a sequence encoding an N-terminal portion of a mouse stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:4 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:4), wherein the sequence encoding the N-terminal portion of the mouse stereocillin protein is attached at its 3' end to an in-frame polynucleotide encoding an N-intein. and b) a second vector, the second vector comprising a polynucleotide comprising a sequence encoding a C-terminal portion of a mouse stereocillin protein (e.g., the C-terminal portion of SEQ ID NO: 4 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 4), wherein the sequence encoding the C-terminal portion of the mouse stereocillin protein comprises at its 5' end an in-frame nucleic acid sequence encoding a C-intein.

[0176] In some embodiments, the sequence encoding the N-terminal portion of the mouse stereocillin protein is a sequence encoding amino acids 1-730 of the stereocillin protein (e.g., amino acids 1-730 of SEQ ID NO: 4), and the sequence encoding the C-terminal portion of the mouse stereocillin protein encodes the remaining amino acids of the stereocillin protein (e.g., amino acids 731-1809 of SEQ ID NO: 4). In this embodiment, the sequence of the N-terminal portion is nucleotides 1-2190 of the polynucleotide encoding mouse stereocillin (e.g., nucleotides 1-2190 of SEQ ID NO: 6), and the sequence of the C-terminal portion is from nucleotide 2191 to the 3' end of the coding sequence of the polynucleotide encoding mouse stereocillin (e.g., the remaining nucleotides of SEQ ID NO: 6, e.g., nucleotides 2191-5430). In some embodiments, the sequence encoding the N-terminal portion of the mouse stereocillin protein is a sequence encoding amino acids 1-746 of the stereocillin protein (e.g., amino acids 1-746 of SEQ ID NO: 4), and the sequence encoding the C-terminal portion of the mouse stereocillin protein encodes the remaining amino acids of the stereocillin protein (e.g., amino acids 747-1809 of SEQ ID NO: 4). In this embodiment, the sequence of the N-terminal portion is nucleotides 1-2238 of the polynucleotide encoding mouse stereocillin (e.g., nucleotides 1-2238 of SEQ ID NO: 6), and the sequence of the C-terminal portion is from nucleotide 2239 to the 3' end of the coding sequence of the polynucleotide encoding mouse stereocillin (e.g., the remaining nucleotides of SEQ ID NO: 6, e.g., nucleotides 2239-5430). In some embodiments, the sequence encoding the N-terminal portion of the mouse stereocillin protein is a sequence encoding amino acids 1 to 969 of the stereocillin protein (e.g., amino acids 1 to 969 of SEQ ID NO: 4), and the sequence encoding the C-terminal portion of the mouse stereocillin protein encodes the remaining amino acids of the stereocillin protein (e.g., amino acids 970 to 1809 of SEQ ID NO: 4).In this embodiment, the sequence of the N-terminal portion is nucleotides 1 to 2907 of the polynucleotide encoding mouse stereocillin (e.g., nucleotides 1 to 2907 of SEQ ID NO: 6), and the sequence of the C-terminal portion is from nucleotide 2908 to the 3' end of the coding sequence of the polynucleotide encoding mouse stereocillin (e.g., the remaining nucleotides of SEQ ID NO: 6, e.g., nucleotides 2908 to 5430). In some embodiments, the sequence encoding the N-terminal portion of the mouse stereocillin protein is a sequence encoding amino acids 1 to 1002 of the stereocillin protein (e.g., amino acids 1 to 1002 of SEQ ID NO: 4), and the sequence encoding the C-terminal portion of the mouse stereocillin protein encodes the remaining amino acids of the stereocillin protein (e.g., amino acids 1003 to 1809 of SEQ ID NO: 4). In this embodiment, the sequence of the N-terminal portion is nucleotides 1 to 3006 of the polynucleotide encoding mouse stereocillin (e.g., nucleotides 1 to 3006 of SEQ ID NO: 6), and the sequence of the C-terminal portion is nucleotide 3007 to the 3' end of the coding sequence of the polynucleotide encoding mouse stereocillin (e.g., the remaining nucleotides of SEQ ID NO: 6, e.g., nucleotides 3007 to 5430). The split sites were selected based on the identification of partial extein sequences within the peptide sequence of the stereocillin protein, since split inteins require the presence of extein amino acid residues that are not excised from the target protein upon full-length protein recombination. For certain split sites (e.g., split sites at amino acids 730 and 1002), a full-length extein sequence can be generated by incorporating a sequence encoding a short peptide into the split site and fusing the short peptide to the STRC protein at the split site, thereby providing the catalytic amino acid residues.

[0177] In some embodiments, both the first vector and the second vector further comprise a promoter sequence, e.g., a STRC promoter sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 including nucleotides 252 to 537 or 35 to 530 of SEQ ID NO:2, or a nucleotide sequence of SEQ ID NO:48) operably linked to the 5' end of the polynucleotide encoding the first fusion protein (the N-terminal portion of the stereocillin protein fused to the N-intein) and / or the 5' end of the polynucleotide encoding the second fusion protein (the C-terminal portion of the stereocillin protein fused to the C-intein). or a functional portion of SEQ ID NO:48 containing nucleotides 280 to 560 of SEQ ID NO:1, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 including nucleotides 252 to 537 or 35 to 530 of SEQ ID NO:2, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of a functional portion of SEQ ID NO:48 containing nucleotides 280 to 560 of SEQ ID NO:48. In some embodiments, the STRC promoter has the sequence of SEQ ID NO: 1 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 1. In some embodiments, the STRC promoter has the sequence of SEQ ID NO: 2 or a portion thereof or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2 or a portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO: 2 that is capable of controlling expression of the STRC gene.In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 252-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 120-537 of SEQ ID NO:2. In some embodiments, the functional portion of SEQ ID NO:2 includes or has the sequence of nucleotides 35-530 of SEQ ID NO:2. In some embodiments, the STRC promoter is a polynucleotide having the sequence of SEQ ID NO:48 or a functional portion thereof, or a variant having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:48 or a functional portion thereof. In some embodiments, the STRC promoter is a functional portion of SEQ ID NO:48 that is capable of controlling expression of the STRC gene. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280-560 of SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 280-564 of SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124-560 of SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 124-564 of SEQ ID NO:48. In some embodiments, the functional portion of SEQ ID NO:48 includes or has the sequence of nucleotides 1-560 of SEQ ID NO:48. In some embodiments, the STRC promoter in the second nucleic acid vector is the same (i.e., has the same nucleotide sequence) as the STRC promoter in the first nucleic acid vector. In some embodiments, the STRC promoter in the second nucleic acid vector has a different nucleotide sequence than the STRC promoter in the first nucleic acid vector.

[0178] In some embodiments, the N-intein and the C-intein are derived from the same intein or split intein gene. Alternatively, the sequences of the N-intein and the C-intein are derived from two different intein genes that can undergo protein trans-splicing to reconstitute the full-length stereocilin protein. In some embodiments, the same gene is from the same organism or different organisms. A commonly used split intein is derived from the DnaE gene from various organisms. In some embodiments, the polynucleotide encoding the stereocilin protein is split into two parts, each of which corresponds to about half of the total coding sequence of the full-length gene, i.e., the N-terminal part and the C-terminal part. The polynucleotide encoding the N-terminal part of the stereocilin is fused at its 3' end in frame with the polynucleotide encoding the N-intein, and the polynucleotide encoding the C-terminal part of the stereocilin is fused at its 5' end in frame with the polynucleotide encoding the C-intein.

[0179] In some embodiments, the first vector and the second vector, when introduced into a cell (e.g., a cell of a subject, such as a subject with sensorineural hearing loss, e.g., DFNB16), generate a first fusion protein and a second fusion protein. In some embodiments, the first fusion protein contains an N-terminal portion of a stereocilin protein fused at its C-terminus to an N-intein. In some embodiments, the second fusion protein contains a C-terminal portion of a stereocilin protein fused at its N-terminus to a C-intein. In some embodiments, the N-intein of the first fusion protein and the C-intein of the second fusion protein selectively combine to generate a third fusion protein, which contains, from N-terminus to C-terminus, an N-terminal portion of a stereocilin protein, an N-intein bound at its C-terminus to a C-intein, and a C-terminal portion of a stereocilin protein. In some embodiments, the N-intein bound to the C-intein can undergo a trans-splicing reaction that excises the N-intein and C-intein and links the C-terminus of the N-terminal portion of the stereocillin protein to the N-terminus of the C-terminal portion.

[0180] The split intein systems described herein can include a split intein encoded by one gene that is subsequently engineered using routine methods to encode two separate intein fragments (e.g., split inteins). In some embodiments, the split intein is encoded by two separate genes.

[0181] The split inteins of the compositions and methods of the disclosure are useful in the detection of cyanobacteria, including, among others, Nostoc punctiforme (Npu), Synechocystis sp. PCC6803 (Ssp), Fischerella sp. PCC9605 (Fsp), Scytonema tolypothrichoides (Sto), Cyanobacteria bacterium SW_9_47_5, Nodularia spumigena (Nsp), Nostoc flagelliforme (Nfl), Crocosphaera watsonii (Cwa) WH8502, Chroococcidiopsis cubana (Ccu) CCALA043, Trichodesmium erythraeum (Ter), Rhodothermus marinus (Rma), Saccharomyces cerevisiae (Sce), Saccharomyces castellii (Sca), Saccharomyces cerevisiae (Sce), Saccharomyces castellii (Sca), Saccharomyces erythraeum (Sca), Saccharomyces cerevisiae ... The DnaE gene may be derived from a DnaE gene (e.g., DNA polymerase III subunit alpha) from Pyrococcus unisporus (Sun), Zygosaccharomyces bisporus (Zbi), Torulaspora pretoriensis (Tpr), Mycobacteria tuberculosis (Mtu), Mycobacterium leprae (Mle), Mycobacterium smegmatis (Msm), Pyrococcus abyssi (Pab), Pyrococcus horikoshii (Pho), Coxiella burnetti (Cbu), Coxiella neoformans (Cne), Coxiella gattii (Cga), Histoplasma capsulatum (Hca), and Porphyra purpurea chloroplast (Ppu). In some embodiments, the split intein is derived from multiple sequence alignment studies of DnaE to identify a consensus design (e.g., Cfa) for engineering a split intein with desired stability and activity (e.g., the split intein is a Cfa intein).Other split intein systems suitable for use with the compositions and methods disclosed herein include those described in International Patent Application Publication Nos. WO2020 / 249723, WO2021 / 099607, and WO2021 / 047558, U.S. Patent Application Publication Nos. US20210371878A1, US20220275027A1, and US20200277333A. No. 1, as well as U.S. Pat. Nos. 10,066,027, 10,526,401, 11,142,550, 11,306,324, 10,100,080, and 8,394,604, each of which is incorporated herein by reference in its entirety with respect to split intein systems.

[0182] In some embodiments, each of the first vector and the second vector further comprises a 5'ITR at its 5' end and a 3'ITR at its 3' end. In some embodiments, the 5'ITR and the 3'ITR are ITRs of AAV. In some embodiments, the ITRs of AAV are ITRs of AAV2.

[0183] In some embodiments, the two-vector split intein system of the disclosure includes: a) a first vector, comprising, from 5' to 3', i) optionally, a 5' ITR (e.g., the 5' ITR of AAV2); ii) a STRC promoter (e.g., a nucleic acid sequence that is at least 85% (e.g., 95% nucleotides) long, and is at least 85% identical to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 that includes nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 that contains nucleotides 280-560 of SEQ ID NO:48, or a functional portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 that includes nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 that contains nucleotides 289-560 of SEQ ID NO:48). (iii) a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of the stereocillin protein of SEQ ID NO:3 or SEQ ID NO:4); iv) a polynucleotide encoding an N-intein; (v) optionally, a poly(A) sequence; and (vi) optionally, a 3' ITR (e.g., the 3' ITR of AAV2), and b) a second vector, comprising from 5' to 3': i) optionally, a 5' ITR (e.g., the 5' ITR of AAV2);ii) STRC promoter (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 48, a functional portion of SEQ ID NO: 2 including nucleotides 252 to 537 or 35 to 530 of SEQ ID NO: 2, or a functional portion of SEQ ID NO: 48 containing nucleotides 280 to 560 of SEQ ID NO: 48, or a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 48, a functional portion of SEQ ID NO: 2 including nucleotides 252 to 537 or 35 to 530 of SEQ ID NO: 2, or a functional portion of SEQ ID NO: 48 containing nucleotides 280 to 560 of SEQ ID NO: 48, which is at least 85% (e.g., at least 86%, 87%, 88%) %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the STRC promoter; iii) a polynucleotide encoding a C-intein; iv) a polynucleotide encoding a C-terminal portion of a stereocillin protein (e.g., the C-terminal portion of the stereocillin protein of SEQ ID NO: 3 or SEQ ID NO: 4); (v) optionally, a poly(A) sequence; and (vi) optionally, a second vector comprising a 3'ITR (e.g., the 3'ITR of AAV2).

[0184] In some embodiments, the two-vector split-intein system of the present disclosure includes a polynucleotide that encodes an N-intein peptide having the amino acid sequence of SEQ ID NO:7, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 7) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes a C-intein peptide having the amino acid sequence of SEQ ID NO:8, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8. VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 8) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes an N-intein peptide having the amino acid sequence of SEQ ID NO:9, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9. CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP (SEQ ID NO: 9) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes a C-intein peptide having the amino acid sequence of SEQ ID NO:10, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10. MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 10) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes a C-intein peptide having the amino acid sequence of SEQ ID NO:11, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11. VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 11) In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9 (e.g., located 3' of a polynucleotide encoding an N-terminal portion of a stereocillin protein), and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:11 (e.g., located 5' of a polynucleotide encoding a C-terminal portion of a stereocillin protein). In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:7, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:8. In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:7, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:10. In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:7, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:11. In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:9, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:8. In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:9, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:10.In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having the amino acid sequence of SEQ ID NO:9, and a second vector that includes a polynucleotide encoding a C-intein polypeptide having the amino acid sequence of SEQ ID NO:11.

[0185] In some embodiments, the two-vector split intein system of the disclosure includes a polynucleotide that encodes an N-intein peptide having the amino acid sequence of SEQ ID NO:12, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12. CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN (SEQ ID NO: 12) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes a C-intein peptide having the amino acid sequence of SEQ ID NO:13, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:13. MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN (SEQ ID NO: 13) In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having the amino acid sequence of SEQ ID NO: 12 (e.g., located 3' to a polynucleotide encoding the N-terminal portion of a stereocillin protein), and a second vector that includes a polynucleotide encoding a C-intein polypeptide having the amino acid sequence of SEQ ID NO: 13 (e.g., located 5' to a polynucleotide encoding the C-terminal portion of a stereocillin protein).

[0186] In some embodiments, the two-vector split intein system of the disclosure includes a polynucleotide that encodes an N-intein peptide having the amino acid sequence of SEQ ID NO:14, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:14. CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP (SEQ ID NO: 14) In some embodiments, the two-vector split-intein system of the disclosure includes a polynucleotide that encodes a C-intein peptide having the amino acid sequence of SEQ ID NO:15, as set forth below, or having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:15. MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 15) In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having the amino acid sequence of SEQ ID NO: 14 (e.g., located 3' to a polynucleotide encoding the N-terminal portion of a stereocillin protein), and a second vector that includes a polynucleotide encoding a C-intein polypeptide having the amino acid sequence of SEQ ID NO: 15 (e.g., located 5' to a polynucleotide encoding the C-terminal portion of a stereocillin protein).

[0187] In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CFSGDTLVALTD (SEQ ID NO: 16). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CLAGDTLITLA (SEQ ID NO: 17). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CLQNGTRLLR (SEQ ID NO: 18). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CLTGDSQVLTR (SEQ ID NO: 19). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CLTYETEIMTV (SEQ ID NO: 20). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of CLSGNTKVRFRY (SEQ ID NO: 21). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding an N-intein peptide having an amino acid sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 16-21.

[0188] In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding a C-intein peptide having an amino acid sequence of GVFVHN (SEQ ID NO: 22). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding a C-intein peptide having an amino acid sequence of GLLVHN (SEQ ID NO: 23). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding a C-intein peptide having an amino acid sequence of GLIASN (SEQ ID NO: 24). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding a C-intein peptide having an amino acid sequence of GLVVHN (SEQ ID NO: 25). In some embodiments, the two-vector split intein system of the present disclosure includes a polynucleotide encoding a C-intein peptide having an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs:22-25.

[0189] In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO: 16 (e.g., located 3' to the polynucleotide encoding the N-terminal portion of a stereocillin protein) and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO: 22 (e.g., located 5' to the polynucleotide encoding the C-terminal portion of a stereocillin protein). In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO: 19 (e.g., located 3' to the polynucleotide encoding the N-terminal portion of a stereocillin protein) and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO: 23 (e.g., located 5' to the polynucleotide encoding the C-terminal portion of a stereocillin protein). In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:20 (e.g., located 3' to the polynucleotide encoding the N-terminal portion of a stereocillin protein) and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:24 (e.g., located 5' to the polynucleotide encoding the C-terminal portion of a stereocillin protein). In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence of SEQ ID NO:21 (e.g., located 3' to the polynucleotide encoding the N-terminal portion of a stereocillin protein) and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence of SEQ ID NO:25 (e.g., located 5' to the polynucleotide encoding the C-terminal portion of a stereocillin protein).

[0190] In some embodiments, the two-vector split intein system of the disclosure collectively includes one or more polynucleotides encoding an N-intein and C-intein pair set forth in Table 5 below, or one or more polynucleotides encoding an N-intein and C-intein pair that have at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to an N-intein and C-intein pair set forth in Table 5. In some embodiments, the two-vector split intein system includes a first vector that includes a polynucleotide encoding an N-intein peptide having an amino acid sequence set forth in Table 5 (e.g., located 3' to a polynucleotide encoding the N-terminal portion of a stereocillin protein), and a second vector that includes a polynucleotide encoding a C-intein polypeptide having an amino acid sequence set forth in the same row as the amino acid sequence of the N-intein in Table 5 (e.g., located 5' to a polynucleotide encoding the C-terminal portion of a stereocillin protein).

[0191] [Table 5-1]

[0192] [Table 5-2]

[0193] [Table 5-3]

[0194] The Npu N-intein of SEQ ID NO:26 can be encoded by a polynucleotide having the DNA sequence of SEQ ID NO:40, shown below. TGCCTGAGCTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGAATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGAGGCGAGC AGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGAGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGAGCTGGACCTGATGAGAGTGGACAACCTGCCCAAC (SEQ ID NO: 40) The Npu C-intein of SEQ ID NO:27 can be encoded by a polynucleotide having the DNA sequence of SEQ ID NO:41, shown below. ATCAAGATCGCCACAAGAAAGTACCTGGGCAAGCAGAACGTGTACGACATCGGCGTGGAGAGAGACCACAACTTCGCCCTGAAGAACGGCTTCATCGCCAGCAAT (SEQ ID NO: 41) Split inteins of the present disclosure (i.e., N-inteins and C-inteins) can include a nucleophilic amino acid at or near its N- or C-terminus that is capable of a trans-splicing reaction, hi some embodiments, the nucleophilic amino acid is selected from serine, threonine, cysteine, or alanine.

[0195] In some embodiments, the first vector and / or the second vector further include one or more additional regulatory sequences, such as, for example, a WPRE sequence, an enhancer sequence, a poly(A) sequence, a terminator sequence, or a degradation signal, among others.

[0196] In some embodiments, the split intein systems described herein include ligand-dependent inteins that effect protein splicing upon contact with a ligand (e.g., a small molecule such as 4-hydroxytamoxifen, a peptide, a protein, a polynucleotide, an amino acid, a nucleotide, etc.) Various ligand-dependent inteins are described in US2014 / 0065711, the disclosure of which is incorporated herein by reference with respect to ligand-dependent inteins.

[0197] The present disclosure provides vectors that contain one or more degradation signals within an intein (e.g., N-intein or C-intein) polypeptide(s) that mediate protein degradation by the ubiquitin-proteasome system and / or the autophagy-lysosomal pathway. Such sequences can be incorporated into the vector systems of the present disclosure to avoid or reduce accumulation of excised intein proteins in target cells. Exemplary degradation signals include N-degrons and C-degrons, which are peptide sequences that contain motifs that contain lysine residues that can be targeted for polyubiquitination and subsequent degradation. In some embodiments, the degron is a degradation signal located within an intein that is not at the N- or C-terminus of the intein. In some embodiments, an N-intein protein contains one or more (e.g., two, three, four, five, or more) degrons. In some embodiments, a C-intein protein contains one or more (e.g., two, three, four, five, or more) degrons. In some embodiments, the degron is a CL1 degron, which is a C-terminal destabilizing peptide that shares structural similarity with misfolded proteins and is recognized by the ubiquitination system. In some embodiments, the degron is a PB29 degron, an SMN degron, a CIITA degron, or an ODC degron. Such degradation signals are described in WO2016 / 13932, which is incorporated herein by reference in connection with degradation signals. Another example of a degradation signal includes a degron derived from E. coli dihydrofolate reductase (ecDHFR), as described in WO2020 / 079034, which is incorporated herein by reference.Additional degradation signals include the FKBP12 degradation domain (Banaszynski et al., Cell 126:995-1004, 2006), the PEST degradation domain (Rechsteiner and Rogers, Trends Biochem Sci. 21:267-271, 1996), the UbR tag ubiquitination signal (Chassin et al., Nat Commun. 10:2013, 2019), and destabilizing mutations in human ELRBD (Miyazaki et al., J. Am. Chem. Soc., 134:3942-3945, 2012).

[0198] Expression of exogenous polynucleotides in mammalian cells Mutations in a wide variety of genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, have been associated with sensorineural hearing loss, and some of these mutations, such as those in MYO7A, have also been associated with vestibular dysfunction. The compositions and methods described herein can be used to detect a polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a STRC promoter operably linked to a polynucleotide sequence encoding a protein of interest (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48). By administering a nucleic acid vector containing a polynucleotide having sequence identity (e.g., a polynucleotide having sequence identity of 0.01 to 0.100, ...

[0199] A protein that can be expressed in the context of the compositions described herein (e.g., a transgene encoding a protein) can be expressed in the context of a STRC promoter (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252 to 537 or 35 to 530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280 to 560 of SEQ ID NO:48) that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, When operably linked to a polynucleotide having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), the polypeptide (when operably linked to a polynucleotide having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) is a protein expressed in healthy hair cells (e.g., cochlear and / or vestibular hair cells, e.g., a protein that plays a role in hair cell development, function, regeneration, cell fate determination, survival, or maintenance, or a protein that is deficient in a subject with sensorineural hearing loss or vestibular dysfunction) or is another protein of therapeutic interest. Proteins that can be expressed in hair cells using the compositions and methods described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D 4, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, GRXCR2, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, ISL1, TMTC4, BIP, and KCNQ4.The STRC promoter described herein can also be used to express short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), components of a gene editing system (e.g., nucleases such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs), or guide RNAs (gRNAs), or microRNAs in hair cells (e.g., cochlear or vestibular hair cells). Additionally, the STRC promoter described herein can be operably linked to a polynucleotide encoding the N-terminal portion of a stereocillin protein (e.g., the N-terminal portion of a wild-type stereocillin protein, e.g., the protein of SEQ ID NO: 3 or SEQ ID NO: 4, etc.) for use in the two-vector system described herein.

[0200] Polynucleotide encoding a protein of interest One platform that can be used to achieve therapeutically effective intracellular concentrations of a protein of interest in mammalian cells is through stable expression of a gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell, or by episomal concatemerization in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of a corresponding protein. To introduce a foreign gene into a mammalian cell, the gene can be incorporated into a vector. The vector can be introduced into the cell by a wide variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are further described in, for example, Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014), and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0201] A target protein can also be introduced into mammalian cells by directing a vector containing a gene encoding the target protein to cell membrane phospholipids. For example, the vector can be directed to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein that has affinity for all cell membrane phospholipids. Such constructs can be generated using methods well known to those skilled in the art.

[0202] The recognition and binding of a polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. Thus, sequence elements may be included within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription initiation site. Such sequence elements include, for example, mammalian promoters, which are sequences that can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters are described in Smith, et al., Mol. Sys. Biol., 3:73 (published online), the disclosure of which is incorporated herein by reference. The promoter used in the methods and compositions described herein is a STRC promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48).

[0203] Once a polynucleotide encoding a protein of interest has been introduced into a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to the mammalian promoter to regulate gene expression. The chemical reagent can function to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing a repressor protein bound to the promoter. Alternatively, the chemical reagent can function to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors such that the rate of transcription of genes located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical reagents that enhance transcription of polynucleotides by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0204] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides containing genes of interest such that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode proteins of interest, and also include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, examples of which include enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (base pairs 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and the RSV enhancer. The enhancer can be spliced ​​into the vector containing the polynucleotide encoding the protein of interest, for example, at the 5' or 3' position relative to the gene. In a preferred orientation, the enhancer is located 5' from the promoter, and thus 5' to the polynucleotide encoding the protein of interest.

[0205] Without being bound by theory, the inventors believe that the nucleotide sequence linking the 3' end of the STRC promoter and the 5' start site (ATG) of the polynucleotide encoding the protein of interest may play a role in the expression of the protein of interest. In some embodiments, the linking sequence includes a Kozak sequence or a portion thereof. In some embodiments, the linking sequence includes a multiple cloning site or a portion thereof that is used to insert the STRC promoter and / or the coding sequence of the protein of interest into the vector.

[0206] Nucleic acid vectors containing the STRC promoter described herein may include a Woodchuck Post-Transcriptional Regulatory Element (WPRE). The WPRE acts at the mRNA level by facilitating nuclear export of the transcript and / or increasing the efficiency of polyadenylation of the nascent transcript, thereby increasing the total amount of mRNA in the cell. The addition of the WPRE to the vector can result in substantial improvement in transgene expression levels from several different promoters, both in vitro and in vivo.

[0207] In some embodiments, the nucleic acid vectors containing the STRC promoter described herein include reporter sequences, which may be useful for confirming expression of genes operably linked to the STRC promoter, for example, in cells and tissues (e.g., cochlear hair cells, such as outer hair cells and inner hair cells, and vestibular hair cells, such as type I and type II vestibular hair cells). Reporter sequences that may be provided within a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When combined with regulatory elements, such as the STRC promoter, that drive their expression, the reporter sequences provide a signal that is detectable by conventional means, for example, enzymatic assays, radioactive assays, colorimetric assays, fluorescent assays or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunoassays such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemical staining. For example, if the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be visually measured by color or light production in a luminometer.

[0208] Methods for delivery of exogenous polynucleotides to target cells Techniques that can be used to introduce a transgene, for example a transgene operably linked to the STRC promoter described herein, into a target cell (e.g., a mammalian cell) are well known in the art. For example, electroporation can be used to permeabilize a mammalian cell (e.g., a human target cell) by applying an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, exposed to an external electric field in this manner are then susceptible to uptake of exogenous polynucleotides. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate uptake of exogenous polynucleotides into eukaryotic cell nuclei. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0209] Further techniques useful for transfection of target cells include squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that it does not require a vector for the delivery of polynucleotides into cells, such as human target cells. Squeeze-poration is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0210] Lipofection is another technique useful for transfection of target cells. This involves loading polynucleotides into liposomes that often present cationic functional groups, such as quaternary amines or protonated amines, toward the liposome exterior. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately resulting in the uptake of exogenous polynucleotides, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous polynucleotides includes contacting cells with cationic polymer-polynucleotide complexes. Exemplary cationic molecules that can be coupled to polynucleotides to impart a positive charge favorable for interaction with cell membranes include activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran (the use of which as a transfection agent is detailed, for example, in Gulick et al., Current Protocols in Molecular Biology 40:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference). Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to guide the uptake of polynucleotides. This technology is detailed, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0211] Another useful tool for inducing the uptake of exogenous polynucleotides by target cells is laserfection, also called optical transfection, a technique that involves exposing cells to electromagnetic radiation of specific wavelengths to gently permeabilize the cells and allow the polynucleotides to penetrate the cell membrane. The biological activity of this technique has been found to be similar to, and in some cases superior to, electroporation.

[0212] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and halowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the genes intended for intracellular delivery is attached to the nanostructure surface. A tip with an array of these needles is then pressed against a cell or tissue. Cells impaled by the nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0213] Magnetofection can also be used to deliver polynucleotides to target cells. The principle of magnetofection is to bind polynucleotides to cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, are fully biodegradable, and are coated with specific cationic proprietary molecules that vary depending on the application. The binding of these particles to gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells under the influence of an external magnetic field generated by a magnet. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0214] Another useful tool for inducing the uptake of exogenous polynucleotide by target cells is sonoporation, which is a technique that involves using sound waves (typically ultrasonic frequencies) to permeabilize cell plasma membrane, so as to make cell permeable and allow polynucleotide to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0215] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and genome-modifying proteins, such as nucleases, can be used to efficiently deliver proteins into cells, which then catalyzes site-specific cleavage of endogenous polynucleotide sequences, preparing the genome of the cell for covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called Gesicle, for genetic modification of eukaryotic cells is detailed, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0216] Vectors for delivering exogenous polynucleotides to target cells In addition to achieving high transcription and translation rates, stable expression of foreign genes in mammalian cells can be achieved by integrating the polynucleotide containing the gene into the nuclear genome of mammalian cells. A wide variety of vectors have been developed for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors for use in the compositions and methods described herein contain a STRC promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to a polynucleotide encoding a desired expression product (e.g., a polynucleotide encoding a protein of interest or a polynucleotide that can be transcribed to produce an inhibitory RNA), as well as additional sequence elements, e.g., used for expression of these substances and / or additional sequence elements used for integration of these polynucleotide sequences into a mammalian cell genome. Vectors that can contain a STRC promoter operably linked to a polynucleotide encoding a desired expression product (e.g., a transgene encoding a protein of interest) include plasmids (e.g., a circular DNA molecule capable of autonomously replicating inside a cell), cosmids (e.g., a pWE vector or a sCos vector), artificial chromosomes (e.g., a human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), and viral vectors.Certain vectors that can be used to express a desired expression product (e.g., a protein of interest) include plasmids that contain regulatory sequences, such as enhancer regions, that direct gene transcription. Other vectors useful for expressing a desired expression product (e.g., a protein of interest) contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides that encode markers for the selection of cells that contain such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0217] Viral Vectors for Polynucleotide Delivery Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery, because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., viral vectors from the Retroviridae family), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses such as adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma virus, avian C virus, mammalian C virus, mammalian B virus, mammalian D virus, oncoretrovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference in connection with viral vectors for use in gene therapy.

[0218] AAV vectors for polynucleotide delivery In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include: (1) a STRC promoter described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48), (2) a sequence to be expressed (e.g., a polynucleotide encoding a heterologous expression product or a portion of a stereocillin protein), and (3) viral sequences that facilitate integration and expression of the sequence to be expressed. The viral sequence may include sequences of AAV required in cis for DNA replication and packaging into virions (e.g., functional ITRs). In a typical application, the expressed sequence encodes a protein that can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate determination, hair cell survival, or hair cell maintenance, or a wild-type form of a hair cell protein that is mutated in a subject with a form of inherited hearing loss or vestibular dysfunction, which may be useful for improving hearing or vestibular function in a subject with a mutation associated with hearing loss, hearing loss, or vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopsia, or balance disorders). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The ITRs of AAV may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs may be those of AAV2.Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated by reference herein with respect to AAV vectors for gene delivery.

[0219] The polynucleotides and vectors described herein (e.g., the STRC promoter operably linked to a transgene encoding a protein of interest or to a polynucleotide encoding a portion of the WT stereocillin protein) can be incorporated into rAAV virions to facilitate the introduction of the polynucleotide or vector into cells. The capsid protein of AAV constitutes the non-nucleic acid portion of the exterior of the virion and is encoded by the AAV cap gene. The cap gene encodes the three viral coat proteins VP1, VP2, and VP3 required for virion assembly. Construction of rAAV virions is described, for example, in US5,173,414, US5,139,941, US5,863,541, US5,869,305, US6,057,152, and US6,376,237, as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference in their entirety with respect to AAV vectors for gene delivery.

[0220] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a wide variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting to hair cells, AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for transduction of the retina may also be used in the methods and compositions described herein. The construction and use of AAV vectors of different serotypes and AAV proteins are described, for example, in Chao et al., Mol. Ther. 2:619 (2000), Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000), Xiao et al., J. Virol. 72:2224 (1998), Halbert et al., J. Virol. 74:1524 (2000), Halbert et al., J. Virol. 75:6615 (2001), and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated by reference herein with respect to AAV vectors for gene delivery.

[0221] Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with capsid genes from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0222] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to promote targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, such as insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include capsid hybrids made by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0223] Pharmaceutical Compositions A STRC promoter as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) can be operably linked to a polynucleotide encoding a desired expression product (e.g., a transgene encoding a protein of interest) or to a polynucleotide encoding a portion of a stereocillin protein and incorporated into a vehicle for administration to a patient, such as a human patient suffering from sensorineural hearing loss and / or vestibular dysfunction. Pharmaceutical compositions containing vectors such as viral vectors containing the STRC promoter described herein operably linked to a polynucleotide encoding a desired expression product can be prepared using methods known in the art.For example, such compositions can be prepared in a desired form, such as a lyophilized formulation or an aqueous solution, using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013), which is incorporated herein by reference).

[0224] Mixtures of nucleic acid vectors (e.g., viral vectors) containing a STRC promoter described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to a polynucleotide encoding a desired expression product or to a polynucleotide encoding a portion of a stereocillin protein can be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms. Suitable dosage forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions (described in US Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation may be sterile and may be fluid to the extent that it can easily pass through a syringe needle. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0225] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that may be used will be known to those of skill in the art in view of the present disclosure. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymph solution. An exemplary synthetic perilymph solution includes 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM HEPE, at a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The person administering will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations will meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0226] Treatment method The compositions described herein may be administered to a subject having or at risk of developing sensorineural hearing loss and / or vestibular dysfunction by a wide variety of routes, including, for example, local administration to the middle or inner ear (e.g., administration into the perilymph or endolymph, e.g., administration into or through the oval window, round window, or semicircular canal (e.g., horizontal semicircular canal), or administration by transtympanic or intratympanic injection, e.g., administration into hair cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route of administration in any given case will depend on the particular composition administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions can be administered once or multiple times (e.g., annually, twice a year, three times a year, bimonthly, monthly, or biweekly). In embodiments relating to a two-vector system, the first and second nucleic acid vectors can be administered simultaneously (e.g., in one composition) or sequentially (e.g., one nucleic acid vector is administered immediately after the other nucleic acid vector, or 5, 10, 15, 20, 25, 30, 45, 1, 2, or more minutes after the first nucleic acid vector). The first and second nucleic acid vectors can have the same capsid or different capsids (e.g., AAV capsids).

[0227] Subjects who may be treated as described herein are those who have or are at risk of developing sensorineural hearing loss and / or vestibular dysfunction (e.g., subjects who have or are at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects having or at risk of developing cochlear hair cell damage (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or age-related damage), subjects having or at risk of developing vestibular hair cell damage (e.g., disease or infection, head trauma, ototoxic drugs, or age-related damage), subjects having or at risk of developing sensorineural hearing loss, hearing loss, or auditory neuropathy, subjects having or at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopsis, or balance disorders), subjects having tinnitus (e.g., tinnitus alone or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects having a genetic mutation associated with hearing loss and / or vestibular dysfunction, or subjects with a family history of hereditary hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the disease associated with damage or loss of hair cells (e.g., cochlear and / or vestibular hair cells) is an autoimmune disease or condition in which an autoimmune response contributes to hair cell damage or cell death. Autoimmune diseases associated with sensorineural hearing loss and vestibular dysfunction include autoimmune inner ear disease (AIED), polyarteritis nodosa (PAN), Cogan's syndrome, relapsing polychondritis, systemic lupus erythematosus (SLE), Wegener's granulomatosis, Sjogren's syndrome, and Behcet's disease. Some infectious conditions, such as Lyme disease and syphilis, can also cause hearing loss and vestibular dysfunction (e.g., by inducing autoantibody production). Viral infections, such as rubella, cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), HSV types 1 and 2, West Nile virus (WNV), human immunodeficiency virus (HIV), varicella zoster virus (VZV), measles, and mumps can also cause hearing loss and vestibular dysfunction.In some embodiments, the subject has or is at risk of developing hearing loss and / or vestibular dysfunction associated with or resulting from loss of hair cells (e.g., cochlear or vestibular hair cells). The STRC2 vector system described herein can be used to treat subjects with STRC mutations (e.g., mutations that reduce stereocillin function or expression, or STRC mutations associated with sensorineural hearing loss or vestibular dysfunction, e.g., mutations that cause nonsyndromic hearing loss, e.g., DFNB16), e.g., subjects with STRC mutations that exhibit symptoms of hearing loss or vestibular dysfunction, and subjects that do not yet exhibit symptoms (e.g., prophylactic treatment of subjects with STRC mutations), or subjects whose STRC mutation status and / or STRC activity level are unknown. The methods described herein can include screening the subject for one or more genetic mutations known to be associated with hearing loss and / or vestibular dysfunction prior to treatment or administration with the compositions described herein. Screening the subject for genetic mutations can be performed using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein can also include assessing hearing and / or vestibular function in a subject prior to treatment or administration with a composition described herein. Hearing can be assessed using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochleogram (ECOG), and otoacoustic emissions. Vestibular function can be assessed using standard tests such as eye movement tests (e.g., electronystagmography (ENG) or video nystagmography (VNG)), vestibulo-ocular reflex (VOR) tests (e.g., head impulse test (Halmagyi-Curthoys test), which can be performed either at the bedside or using video head impulse testing (VHIT), or caloric reflex test), stabilography, rotary-chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), and balance tests in specialized clinics such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010).These tests can also be used to assess hearing and / or vestibular function in a subject after treatment or administration with the compositions described herein. The compositions and methods described herein can also be administered as prophylactic treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, such as patients with a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), patients with genetic mutations associated with hearing loss or vestibular dysfunction who have not yet demonstrated hearing loss or vestibular dysfunction, or patients who have been exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., disease or infection, head trauma, ototoxic drugs, or aging). The compositions and methods described herein can also be used to treat subjects with idiopathic vestibular dysfunction.

[0228] The compositions and methods described herein can be used to induce or increase hair cell regeneration in a subject (e.g., cochlear and / or vestibular hair cell regeneration). Subjects who may benefit from compositions that induce or increase hair cell regeneration include those suffering from hearing loss or vestibular dysfunction as a result of hair cell loss (e.g., hair cell loss associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and those with abnormal hair cells (e.g., hair cells that do not function properly compared to normal hair cells), damaged hair cells (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or reduced hair cell numbers due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., to increase survival of damaged hair cells, promote repair of damaged hair cells, or preserve hair cells in subjects at risk for hair cell loss (e.g., hair cell loss due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs). The compositions and methods described herein can also be used to promote or increase hair cell maturation, improve hair cell structure (e.g., improve stereocilia bundle morphology), or improve hair cell function (e.g., improve stereocilia bundle tilt), which can result in improved hearing and / or vestibular function.

[0229] The compositions and methods described herein can also be used to prevent or reduce hearing loss and / or vestibular dysfunction caused by ototoxic drug-induced hair cell damage or cell death (e.g., cochlear and / or vestibular hair cell damage or cell death) in subjects who have been treated with, or are currently undergoing, or about to begin treatment with, an ototoxic drug. Ototoxic drugs are toxic to cells of the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular disorders (bilateral vestibular hypofunction), or oscillopia), tinnitus, or a combination of these symptoms. Drugs known to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic agents (e.g., platinum-containing chemotherapy agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially at high doses), and quinine. In some embodiments, the methods and compositions described herein can be used to treat bilateral vestibular disorders (bilateral vestibular hypofunction) or oscillopsia. Bilateral vestibular disorders (bilateral vestibular hypofunction) and oscillopsia can be induced by aminoglycosides (e.g., the methods and compositions described herein can be used to promote or increase hair cell regeneration in subjects with or at risk of developing aminoglycoside-induced bilateral vestibular disorders (bilateral vestibular hypofunction) or oscillopsia).

[0230] A polynucleotide encoding a desired expression product operably linked to a STRC promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) for treatment of a subject as described herein can be used to encode a protein expressed in healthy hair cells (e.g., a protein that is involved in hair cell development, hair cell function ... The transgene may be a transgene encoding a protein that plays a role in cell fate determination, hair cell regeneration, hair cell survival, or hair cell maintenance, or a protein that is deficient in a subject with sensorineural hearing loss or vestibular dysfunction, a transgene encoding another protein of interest (e.g., a reporter protein such as a fluorescent protein, lacZ, or luciferase), or a polynucleotide that can be transcribed to produce an shRNA, an ASO, a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA. The transgene can be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss or vestibular dysfunction is associated with a particular genetic mutation, the transgene can be a wild-type form of the gene that is mutated in the subject, and if the subject has hearing loss and / or vestibular dysfunction associated with hair cell loss, the transgene can encode a protein that promotes hair cell regeneration), the severity of the subject's hearing loss or vestibular dysfunction, the health of the subject's hair cells, the subject's age, the subject's family history of hearing loss or vestibular dysfunction, or other factors.Proteins that may be expressed by a transgene operably linked to the STRC promoter for treatment of a subject as described herein include, but are not limited to, ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CI B2, CACNA2D4, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, GRXCR2, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, ISL1, TMTC4, BIP, and KCNQ4. A polynucleotide encoding the N-terminal portion of stereocillin can also be operably linked to the STRC promoter described herein (e.g., in the first vector of a two-vector system). In some embodiments, a polynucleotide encoding a fusion protein containing a C-intein and a C-terminal portion of a stereocilin can be operably linked to a STRC promoter described herein (e.g., in the case of a second vector of an intein expression system).

[0231] Treatment may include administration of various unit doses of a composition containing a nucleic acid vector (e.g., AAV vector) containing the STRC promoter described herein. Each unit dose typically contains a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route of administration and formulation, are within the skill of those skilled in the clinical field. The unit dose need not be administered in a single injection, but may include continuous infusion over a set period of time. Administration may be performed using a syringe pump to control the rate of infusion to minimize damage to the inner ear (e.g., the cochlea and / or vestibular system). When the nucleic acid vector is an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector), the viral vector can be, for example, 1 μL to 200 μL (e.g., 1 μL to 200 μL). L, 2μL, 3μL, 5μL, 6μL, 7μL, 8μL, 9μL, 10μL, 15μL, 20μL, 25μL, 30μL, 35μL, 40μL, 45μL, 50μL, 55μL, 60μL, 65μL, 70μL, 75μL , approximately 1 9 Vector genome (VG) / mL ~ approx. 1 x 10 16 VG / mL (e.g., 1×10 9 VG / mL, 2×10 9 VG / mL, 3×10 9 VG / mL, 4×10 9 VG / mL, 5×10 9 VG / mL, 6×10 9 VG / mL, 7×10 9 VG / mL, 8×10 9 VG / mL, 9×10 9 VG / mL, 1×10 10 VG / mL, 2×10 10 VG / mL, 3×10 10 VG / mL, 4×10 10VG / mL、5×10 10 VG / mL、6×10 10 VG / mL、7×10 10 VG / mL、8×10 10 VG / mL、9×10 10 VG / mL、1×10 11 VG / mL、2×10 11 VG / mL、3×10 11 VG / mL、4×10 11 VG / mL、5×10 11 VG / mL、6×10 11 VG / mL、7×10 11 VG / mL、8×10 11 VG / mL、9×10 11 VG / mL、1×10 12 VG / mL、2×10 12 VG / mL、3×10 12 VG / mL、4×10 12 VG / mL、5×10 12 VG / mL、6×10 12 VG / mL、7×10 12 VG / mL、8×10 12 VG / mL、9×10 12 VG / mL、1×10 13 VG / mL、2×10 13 VG / mL、3×10 13 VG / mL、4×10 13 VG / mL、5×10 13 VG / mL、6×10 13 VG / mL、7×10 13 VG / mL、8×10 13 VG / mL、9×10 13 VG / mL、1×10 14 VG / mL、2×10 14 VG / mL、3×10 14 VG / mL、4×10 14 VG / mL、5×10 14 VG / mL、6×10 14 VG / mL、7×10 14 VG / mL、8×10 14 VG / mL、9×10 14 VG / mL、1×10 15 VG / mL、2×10 15 VG / mL、3×1015 VG / mL, 4×10 15 VG / mL, 5×10 15 VG / mL, 6×10 15 VG / mL, 7×10 15 VG / mL, 8×10 15 VG / mL, 9×10 15 VG / mL, or 1×10 16 The AAV vector may be administered to a patient at a dose of approximately 1×10 7 VG / ear ~ approx. 2 x 10 15 VG / ear (e.g. 1×10 7 VG / ear, 2×10 7 VG / ear, 3×10 7 VG / ear, 4×10 7 VG / ear, 5×10 7 VG / ear, 6×10 7 VG / ear, 7×10 7 VG / ear, 8×10 7 VG / ear, 9×10 7 VG / ear, 1×10 8 VG / ear, 2×10 8 VG / ear, 3×10 8 VG / ear, 4×10 8 VG / ear, 5×10 8 VG / ear, 6×10 8 VG / ear, 7×10 8 VG / ear, 8×10 8 VG / ear, 9×10 8 VG / ear, 1×10 9 VG / ear, 2×10 9 VG / ear, 3×10 9 VG / ear, 4×10 9 VG / ear, 5×10 9 VG / ear, 6×10 9 VG / ear, 7×10 9 VG / ear, 8×10 9 VG / ear, 9×10 9 VG / ear, 1×10 10 VG / ear, 2×10 10 VG / ear, 3×10 10 VG / ear, 4×10 10 VG / ear, 5×10 10 VG / ear, 6×10 10 VG / ear, 7×10 10VG / ear, 8×10 10 VG / ear, 9×10 10 VG / ear, 1×10 11 VG / ear, 2×10 11 VG / ear, 3×10 11 VG / ear, 4×10 11 VG / ear, 5×10 11 VG / ear, 6×10 11 VG / ear, 7×10 11 VG / ear, 8×10 11 VG / ear, 9×10 11 VG / ear, 1×10 12 VG / ear, 2×10 12 VG / ear, 3×10 12 VG / ear, 4×10 12 VG / ear, 5×10 12 VG / ear, 6×10 12 VG / ear, 7×10 12 VG / ear, 8×10 12 VG / ear, 9×10 12 VG / ear, 1×10 13 VG / ear, 2×10 13 VG / ear, 3×10 13 VG / ear, 4×10 13 VG / ear, 5×10 13 VG / ear, 6×10 13 VG / ear, 7×10 13 VG / ear, 8×10 13 VG / ear, 9×10 13 VG / ear, 1×10 14 VG / ear, 2×10 14 VG / ear, 3×10 14 VG / ear, 4×10 14 VG / ear, 5×10 14 VG / ear, 6×10 14 VG / ear, 7×10 14 VG / ear, 8×10 14 VG / ear, 9×10 14 VG / ear, 1×10 15 VG / ear, or 2×10 15 The subject may be administered a dose of 100 mg / ear (VG / ear).

[0232] The compositions described herein are intended to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, treat bilateral vestibular disorders, treat oscillopia, treat balance disorders, increase or induce expression in hair cells of a protein encoded by a transgene operably linked to the STRC promoter, increase function in hair cells of a protein encoded by a transgene operably linked to the STRC promoter, prevent or reduce hair cell damage (e.g., hair cell damage associated with acoustic trauma, head trauma, ototoxic drugs, disease or infection, or aging). the hair cell death (e.g., ototoxic drug-induced hair cell death, noise-associated hair cell death, aging-associated hair cell death, disease or infection-associated hair cell death, or head trauma-associated hair cell death), promote or increase hair cell generation, increase hair cell number, induce or increase hair cell regeneration (e.g., cochlear and / or vestibular hair cell regeneration), promote or increase hair cell survival, promote or increase hair cell maturation, improve hair bundle attachment to the tectorial membrane (e.g., OHC hair bundle attachment), improve hair cell structure, or improve hair cell function. Hearing can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleogram (ECOG), and otoacoustic emissions) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hearing measurements obtained before treatment.Vestibular function can be assessed using standard tests for balance and vertigo, such as eye movement tests (e.g., ENG or VNG), stabilometry, VOR testing (e.g., head impulse test (Halmagyi-Curthoys test, e.g., VHIT), or caloric reflex testing, rotary-chair testing, and The ability of a subject to understand speech may be assessed using a variety of methods, including, but not limited to, neuropsychological testing, ECOG, VEMP, and outpatient balance testing, and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to measurements taken prior to treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered to subjects (e.g., subjects with a genetic mutation associated with hearing loss or vestibular dysfunction who have a family history of hearing loss or vestibular dysfunction (e.g., inherited hearing loss or vestibular dysfunction), or subjects who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma) but have not developed a hearing loss or vestibular dysfunction (e.g., The composition may be administered in an amount sufficient to slow or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction in subjects who do not exhibit symptoms such as dizziness, vertigo, dizziness, or imbalance, or in subjects who exhibit mild to moderate hearing loss or vestibular dysfunction. Expression of the protein encoded by the transgene operably linked to the STRC promoter in the nucleic acid vector administered to the subject may be assessed using immunohistochemical staining, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting proteins or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to expression before administration of the compositions described herein.The number of hair cells, the function of hair cells, the regeneration of hair cells, or the function of a protein encoded by a transgene operably linked to a STRC promoter in a nucleic acid vector administered to a subject may be indirectly assessed based on a hearing test or a test of vestibular function, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to the number of hair cells, the function of hair cells, the regeneration of hair cells, or the function of the protein before administration of a composition described herein. Hair cell damage or cell death may be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hair cell damage and cell death typically observed in untreated subjects. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more after administration of the compositions described herein. Patients may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more after administration of the compositions, depending on the dose and route of administration used for treatment. Depending on the results of the evaluation, patients may receive additional treatment.

[0233] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss and / or vestibular dysfunction. The compositions can include a STRC promoter as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48), a nucleic acid vector containing such a polynucleotide, a nucleic acid vector containing a STRC promoter as described herein operably linked to a polynucleotide encoding a desired expression product (e.g., a transgene encoding a protein of interest, e.g., a protein expressible in a hair cell to treat hearing loss and / or vestibular dysfunction), as well as a nucleic acid vector system that includes a STRC promoter as described herein (e.g., a two-vector system as described herein). The nucleic acid vector may be packaged in an AAV viral capsid (e.g., AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B). The kit may further include a package insert instructing a user of the kit, such as a physician, to practice the methods described herein. The kit optionally includes a syringe or other device for administering the composition. EXAMPLES

[0234] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0235] Example 1. STRC promoter-driven GFP expression is enriched in outer hair cells of the organ of Corti and restricted to hair cells of the utricle in mice To test the specificity of the STRC promoter disclosed herein in vivo, AAV vectors containing eGFP operably linked to several of the STRC promoters disclosed herein were injected into wild-type C57BL / 6J mice. Using well-known methods for AAV vector preparation, an AAV vector expressing eGFP under the control of the STRC promoter of SEQ ID NO:1 was prepared from transgene plasmid P1208 (FIG. 1). Using well-known methods for AAV vector preparation, an AAV vector expressing eGFP under the control of the STRC promoter of nucleotides 35-530 of SEQ ID NO:2 was prepared from transgene plasmid P1209 (FIG. 2).

[0236] Early postnatal (2-3 days after birth) mice were injected with one of two AAV vectors in 1 μl via a fenestration in the posterior semicircular canal (1.6 × 10 for the AAV vector expressing eGFP under the control of the STRC promoter at nucleotides 35-530 of SEQ ID NO:2). 10 vg / ear; 1.1 × 10 for AAV vector expressing eGFP under the control of the STRC promoter of SEQ ID NO:1 10 vg / ear).

[0237] Six-week-old adult mice were injected with one of two AAV vectors in 2 μl via a fenestration in the posterior semicircular canal (3.2 × 10 for the AAV vector expressing eGFP under the control of the STRC promoter at nucleotides 35 to 530 of SEQ ID NO:2). 10 vg / ear; 2.2 × 10 for AAV vector expressing eGFP under the control of the STRC promoter of SEQ ID NO:1 10 vg / ear).

[0238] After 4 weeks of age, neonatally injected animals were sacrificed and fixed in 10% NBF via cardiac perfusion, and their temporal bones were harvested and kept in 10% NBF for an additional 16 hours. For adult mice, animals were sacrificed 2 weeks after injection and temporal bones were harvested using the same procedure. After 2 days of decalcification in 8% EDTA, ears were microdissected and the utricle and organ of Corti were prepared for immunohistochemical staining. Whole mount preparations of the organ of Corti were counterstained with Myosin7a antibody to visualize all hair cells (Fig. 3, panels A and A'; Fig. 4, panels A, A', and A''; Fig. 7, panels A and A'; Fig. 8, panels A, A', and A'') and imaged under a confocal microscope (Leica SP8, 20x / 0.75 NA, 2 μm step size at 2 AU) along with virus-mediated wild-type GFP expression (Fig. 3, panels B and B'; Fig. 4, panels B, B', and B''; Fig. 7, panels B and B'; Fig. 8, panels B, B', and B'').

[0239] Whole mount preparations of neonatal utricles were counterstained with Pou4f3 antibody to label all hair cell nuclei (Fig. 5, panels A, A' and A'') and Sox2 antibody to label all supporting cells and type II hair cell nuclei (Fig. 5, panels C, C' and C'') and imaged under confocal microscopy (Leica SP8 20x / 0.75 NA, 1.5 μm step size at 2 AU) along with virus-mediated wild-type GFP expression (Fig. 5, panels B, B' and B''). We found that STRC promoter-induced GFP expression was significantly enriched in outer hair cells of the organ of Corti and restricted to hair cells in the utricle. Faint GFP expression was also detected in some inner hair cells of the organ of Corti.

[0240] To visualize stereocillin wild-type expression in mice, adult CBA / CaJ mice were sacrificed and fixed in 10% NBF via cardiac perfusion, and their temporal bones were harvested and kept in 10% NBF for an additional 16 hours. After decalcification in 8% EDTA for 2 days, the organ of Corti was microdissected and stained with an antibody against stereocillin and counterstained with phalloidin, which labels filamentous actin in the stereocilia. Images were acquired using a confocal microscope (Zeiss LSM800, 63x / 1.4 NA, 0.38 μm step size at 1 AU) (Figure 6).

[0241] Example 2. STRC promoter-driven GFP expression is enriched in outer hair cells of the organ of Corti and restricted to hair cells in non-human primates To test the specificity of the STRC promoter disclosed herein in vivo, AAV vectors containing nuclear-directed H2B-eGFP operably linked to several of the STRC promoters disclosed herein were injected into the ears of non-human primates (Macaca fascicularis). Using well-known methods for AAV vector preparation, AAV vectors expressing nuclear H2B-eGFP under the control of the STRC promoter of SEQ ID NO:1 were prepared from transgene plasmid P1016 (FIG. 11). Adult non-human primates were injected with 40 μl of vector (3.41×10 for the AAV vector expressing H2B-eGFP under the control of the STRC promoter of SEQ ID NO:1). 13 vg / ml was performed via the round window membrane, and a fenestration of the lateral semicircular canal allowed for outflow of perilymph during the procedure).

[0242] After 4 weeks of age, animals were sacrificed and fixed in 10% NBF via cardiac perfusion, and their temporal bones were harvested and kept in 10% NBF for an additional 4–10 days. After 6 weeks of decalcification in 14% EDTA, ears were either microdissected and the cyst and organ of Corti were prepared for immunohistochemical staining, or ears were decalcified in formic acid for 6 days, paraffin embedded, and sectioned in 5 μm sections.

[0243] Whole-mount preparations of the organ of Corti were counterstained with Pou4f3 antibody to visualize all hair cells (Figure 9, panel A) and imaged under a confocal microscope (Zeiss LSM880, 40× / NA 0.95, 1 μm step size at 2 AU) with virus-mediated GFP expression counterstained with an antibody against GFP (Figure 9, panel B).

[0244] Sections were labeled with an antibody against GFP and stained with a secondary antibody conjugated to alkaline phosphatase, resulting in red staining due to the reaction of the Fast Red dye with the alkaline phosphatase of the secondary antibody. Sections were counterstained blue with hematoxylin to visualize all nuclei, imaged with a color camera at 20x magnification, and converted to grayscale (Figure 10, panel A). To visualize the red signal of the colored anti-GFP staining, the image processing software GIMP was used to extract all blue (hematoxylin) color from the color image using the color selection tool and then converted to grayscale. Only nuclei with red signal nuclear H2B-GFP remained visible (Figure 10, panel B).

[0245] Example 3. Anc80-CMV-mStrc duplicated double vector system restores hearing in stereocillin-deficient mice Using CRISPR-Cas9 technology, we generated stereocillin-deficient mice in the CBA / CaJ background by creating a frameshift at base pair 232 of STRC. Wild-type animals in the CBA / CaJ background showed clear stereocillin antibody staining in the tips of the stereocilia of outer hair cells (OHCs) (Figure 12A, lower panel), whereas 232 bp Strc-KO animals lacked antibody signal (Figure 12B, lower panel). Mouse STRC was encapsulated in a double Anc80 vector, where the first vector carried the CMV promoter and nucleotides 1-3200 of mouse STRC cDNA, and the second vector carried amino acids 2201-5430, generating a 1000 bp overlap between the two full-length cDNAs. After delivery of both vectors at a concentration of 1E10 vg / ear via the posterior semicircular canal into the cochleae of early postnatal 232bp Strc-KO mice, we could observe de novo expression of stereocillin protein in the tips and inner hair cell bodies of OHCs in the organ of Corti of treated 232bp Strc-KO mice (Figure S1C, lower panel).Four weeks after treatment with Anc80-CMV-mStrc(duplicate), we assessed distortion product otoacoustic emissions (DPOAEs) as a direct readout of OHC function, and measured auditory brainstem responses (ABRs) as a measure of intact auditory ascending pathways. Untreated contralateral ears of 232bp Strc-KO animals ("untreated ear") showed a near absence of DPOAEs and highly elevated ABR thresholds, indicative of loss of function of OHCs (Figure 13A and Figure 13B, open circles), whereas treated 232bp Strc-KO animals ("treated hom") showed recovery of hearing thresholds (Figure 13A and Figure 13B, filled circles). The best responder of treated 232bp Strc-KO animals (Figure 13A and Figure 13B, filled squares) showed hearing thresholds close to those of wild type ("CBA / CaJ") (Figure 13A and Figure 13B, triangles). A higher percentage of OHCs in 232bp Strc-KO mice expressing stereocillin after treatment with AAV-Anc80-CMV-mStrc was found to promote hearing recovery (Figure 13C).

[0246] Example 4.2 The vector split-intein system reconstituted full-length stereocillin in vitro To generate the experimental plasmids, DNA encoding amino acids 1-746 of stereocillin ("N-Strc") was engineered fused to DNA encoding the Npu N-intein fragment (encoding the Npu N-intein of SEQ ID NO:26, SEQ ID NO:40) and cloned into a plasmid containing a constitutively active CMV promoter to generate CMV.N-Strc-N-Npu. DNA encoding amino acids 747-1809 of stereocillin ("C-Strc") was engineered fused downstream of DNA encoding the Npu C-intein fragment (encoding the Npu C-intein of SEQ ID NO:27, SEQ ID NO:41) and cloned into a plasmid containing a CMV promoter to generate CMV.C-Npu-C-Strc. As a control, the full-length stereocillin coding sequence ("FL-Strc") was also cloned into the CMV plasmid to generate CMV.FL-Strc. CMV.GFP was used as a negative control.

[0247] HEK293T cells were transfected with either the control plasmid or a combination of N-Strc and C-Strc plasmids using Lipofectamine 3000 kit (Life Technologies) and incubated for 3 days under standard cell culture conditions. Cell cultures were rinsed with PBS, cells were lysed, and proteins were extracted. Protein lysate concentrations were measured using a BCA assay, and constant masses of protein were loaded for Western blotting using antibodies against beta-actin and stereocillin. Densitometric measurements of protein band intensity were used to determine the relative amount of full-length stereocillin from the samples.

[0248] As shown in Figure 14, A and B, the intein designs tested produced full-length stereoisomer bands. Example 5. Evaluation of stereocillin protein expression using different dual vector systems AAV-DJ-CMV-mSTRC double hybrid vectors were generated with different STRC split sites. The 5' and 3' vectors were separated at nucleotide positions 1800, 2247, 2310, 2421, or 2588 (5' vectors contained nucleotides 1-1800, 1-2247, 1-2310, 1-2421, or 1-2588 of mouse STRC, and 3' vectors contained the remaining nucleotides of mouse STRC coding sequence, e.g., nucleotides 1801-5430, 2248-5430, 2311-5430, 2422-5430, or 2589-5430). The recombinogenic region used in the double hybrid vectors was the AP gene fragment. A duplicated double vector system in which the 5' and 3' vectors contained the 1000 common nucleotides of mouse stereocillin (the 5' vector carried nucleotides 79 to 3278 of NM_080459 (corresponding to nucleotides 1 to 3200 of SEQ ID NO:6) and the 3' vector carried nucleotides 2279 to 5508 (corresponding to nucleotides 2201 to 5430 of SEQ ID NO:6)) was also tested.

[0249] HEK293T cells were seeded in tissue culture plates. After the cells attached, AAV was added to the cultures at a multiplicity of infection (MOI) of 1x10^7 vector genomes per cell (5' and 3' vectors were added in a 1:1 ratio). GFP control and full-length STRC control were treated with Lipofectamine 3000 containing a DNA plasmid encoding the transgene instead of being treated with AAV. Treated cells were incubated under standard cell culture conditions for 3 days, after which the cells were lysed and total protein was harvested. 15 μg of lysate for each sample was loaded onto a 3-8% Tris-Acetate / SDS gel and a standard Western blot was performed. To specifically detect stereocillin protein or actin, the blotted membrane was treated with antibodies. To obtain a semi-quantitative indication of expression, densitometry of the detected bands was performed and the signal intensity ratio between stereocillin detection and actin detection was used to estimate the expression amount of stereocillin protein (Figure 15).

[0250] Example 6. STRC promoter-driven GFP expression in neonatal mouse cochlear explants To test the specificity of the STRC promoters disclosed herein in neonatal cochlear hair cells, sensory epithelium was dissected from P0-P2 mice and plated one or two dishes onto MatTek 35 mm dishes with Matrigel-treated #0 10 mm coverslips. 150-200 μL of DMEM + 10% FBS + 10 μg / mL ciprofloxacin was added to each dish. After 1 hour of incubation at 37°C / 5% CO2, AAV vectors expressing nuclear-targeted eGFP-H2B fusions under the control of various STRC promoters disclosed herein (see Table 6 below for details) were added to each dish at 1 x 10 11 vg of IgG. The explants were then incubated for 2 days at 37°C / 5% CO2. After 2 days, the virus-containing medium was removed and replaced with fresh virus-free medium. The explants were then incubated for another 3 days and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature.

[0251] After incubation with PFA, the explants were washed three times with PBS and then incubated in 10% normal donkey serum (NDS) in PBS for 20 min. NDS was removed and the explants were incubated with primary antibody against Myo7a (a cochlear hair cell marker) (diluted 1:1000 in PBS) overnight at 4°C. The next day, the explants were washed three times with PBS and then incubated with fluorescently labeled secondary antibody (diluted 1:1000 in PBS) for 2-3 h at room temperature. After incubation in the secondary antibody, the explants were washed five times with PBS and mounted on microscope slides using Fluoromount mounting medium. Slides were then imaged using a Zeiss Upright Apotome light microscope for 20× images and a Zeiss LSM 880 confocal microscope for 40× images. Representative examples of 40× images are shown in Figures 16A and 16B.

[0252] [Table 6]

[0253] Quantification of cells expressing GFP after infection was performed using Imaris software to analyze 20x images of the whole cochlea acquired with the Upright Apotome. To quantify the total number of hair cells, the Spot function of Imaris software was used to identify Myo7a + The number of cells was quantified. These cells were then filtered by the intensity of the GFP channel. The filter threshold was manually set to select only spots with Myo7a and GFP. This gave the total number of GFP+ hair cells in each sample. The results are shown in Figure 17.

[0254] To quantify the number of inner and outer hair cells, Myo7a+ spots representing inner and outer hair cells were manually isolated. These spots were then filtered by the intensity of the GFP signal to separately quantify the number of GFP+ inner and outer hair cells. The results are shown in Figure 18.

[0255] Example 7. Administration of a composition containing a STRC promoter-containing nucleic acid vector to a subject with sensorineural hearing loss According to the methods disclosed herein, a practitioner of the art can treat a patient, e.g., a human patient, with hearing loss (e.g., sensorineural hearing loss) to improve or restore hearing. To this end, a practitioner of the art can administer to a human patient a composition comprising a STRC promoter as described herein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, e.g., SEQ ID NO:35-530 of SEQ ID NO:2, or SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48) operably linked to a polynucleotide encoding a desired expression product (e.g., a transgene encoding a protein that plays a role in hair cell development, regeneration, cell fate determination, maintenance, function, or survival, or a wild-type version of a gene associated with hearing loss that is mutated in the subject). A composition containing an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion of No. 48 can be administered. A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or through the round window membrane) to treat sensorineural hearing loss.

[0256] After administering the composition to the patient, a physician skilled in the art can monitor the patient's improvement in response to treatment by a variety of methods.For example, the physician can monitor the patient's hearing by performing standard tests such as hearing test, ABR, electrocochleogram (ECOG), and otoacoustic emission after administering the composition.The finding that the patient shows improved hearing in one or more tests after administering the composition compared to the hearing test results before administering the composition indicates that the patient is responding well to treatment.Subsequent doses can be determined and administered as necessary.

[0257] Example 8. Administration of a composition containing a nucleic acid vector containing a STRC promoter to a subject with vestibular dysfunction According to the methods disclosed herein, a practitioner of the art can treat a patient, e.g., a human patient, with vestibular dysfunction (e.g., bilateral vestibular dysfunction) to improve or restore vestibular function (e.g., improved balance or reduced falls). To this end, a practitioner of the art can administer to a human patient a composition comprising a STRC promoter as described herein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, e.g., a functional portion of SEQ ID NO:2 containing nucleotides 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 ...80-560 of SEQ ID NO:48) operably linked to a polynucleotide encoding a desired expression product (e.g., a transgene encoding a protein that plays a role in hair cell development, regeneration, cell fate determination, maintenance, function, or survival, or a wild-type version of a gene associated with vestibular dysfunction that is mutated in the subject). A composition containing an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion of sequence number 48 can be administered. A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into a semicircular canal, such as the horizontal canal), to treat vestibular dysfunction.

[0258] After administering the composition to the patient, the practitioner in the art can monitor the patient's improvement in response to treatment by a variety of methods.For example, the practitioner can monitor the patient's vestibular function by performing standard tests, such as electronystagmus, video nystagmus, rotation test, VOR test, vestibular evoked myogenic potential test, or computerized dynamic stabilization test.The observation that the patient shows improvement in vestibular function in one or more tests after administering the composition compared to the test results obtained before administering the composition indicates that the patient is responding well to treatment.Subsequent doses can be determined and administered as necessary.

[0259] Example 9. Administration of a composition containing a two-vector system containing a STRC promoter operably linked to a stereocillin coding sequence to a subject with sensorineural hearing loss In accordance with the methods disclosed herein, a practitioner of the art can treat a patient, e.g., a human patient, with sensorineural hearing loss (e.g., sensorineural hearing loss associated with a mutation in STRC, such as DFNB16) to improve or restore hearing. To this end, a practitioner of the art can administer to a human patient a composition comprising a STRC promoter operably linked to a STRC transgene (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, e.g., nucleotides 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48, having at least 85% sequence identity (e.g., 86 In one embodiment, compositions can be administered that contain a two-vector nucleic acid expression system, such as a system utilizing two AAV vectors (e.g., an AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B vector) that collectively comprise a polynucleotide having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the AAV vector.

[0260] The two-vector system may be an overlapping dual vector system containing a first AAV vector and a second AAV vector. The overlapping dual vector system may include a first AAV vector including a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO: 3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 3), and a second AAV vector including a polynucleotide encoding a C-terminal portion of a stereocillin protein, where the 3' end of the stereocillin coding sequence of the first vector overlaps with the 5' end of the stereocillin coding sequence of the second vector. In another example, the two-vector system may be a trans-splicing dual vector system containing a first AAV vector and a second AAV vector. The trans-splicing dual vector system may include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3) and a second AAV vector that includes a splice acceptor signal sequence 5' of a polynucleotide encoding a C-terminal portion of a stereocillin protein. In another example, the two-vector system may be a dual hybrid vector system containing a first AAV vector and a second AAV vector.The dual hybrid vector system may include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3), a splice donor signal sequence 3' of the polynucleotide, and a first recombinogenic region 3' of the splice donor signal sequence, and a second AAV vector that includes a polynucleotide encoding a second recombinogenic region, a splice acceptor signal sequence 3' of the recombinogenic region, and a C-terminal portion of a stereocillin protein 3' of the splice acceptor signal sequence. In yet another example, the two-vector system can be a split intein trans-splicing system that includes a first AAV vector and a second AAV vector. A split intein trans-splicing two vector system can include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3) and a polynucleotide encoding an N-terminal intein (N-intein) 3' to such polynucleotide, and a second AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding a C-terminal intein (C-intein) and a polynucleotide encoding a C-terminal portion of a stereocillin protein 3' to such polynucleotide.The above two-vector system may additionally include regulatory sequences, such as enhancers, poly(A) sequences, and STRC untranslated regions (UTRs, e.g., 5'UTR and / or 3'UTR), that are not part of the promoter described herein.

[0261] A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or through the round window membrane) to treat sensorineural hearing loss.

[0262] After administration of the composition to the patient, a practitioner of the art can monitor the expression of the therapeutic protein encoded by the transgene and the improvement of the patient in response to the treatment by a variety of methods. For example, the practitioner can monitor the patient's hearing by performing standard tests such as hearing tests, ABR, electrocochleogram (ECOG), and otoacoustic emissions after administration of the composition. The finding that the patient shows improved hearing in one or more tests after administration of the composition compared to the hearing test results before administration of the composition indicates that the patient is responding well to the treatment. Subsequent doses can be determined and administered as necessary.

[0263] Example 10. Administration of a composition containing a two-vector system containing a STRC promoter operably linked to a stereocillin coding sequence to a subject with vestibular dysfunction. In accordance with the methods disclosed herein, a practitioner of the art can treat a patient, e.g., a human patient, suffering from a vestibular dysfunction (e.g., a vestibular dysfunction associated with a mutation in STRC, e.g., vertigo, dizziness, imbalance, bilateral vestibular dysfunction, oscillopsia, or balance disorders, etc.) to improve vestibular function. To this end, a practitioner of the art can administer to a human patient a composition comprising a STRC promoter operably linked to a STRC transgene (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:48, a functional portion of SEQ ID NO:2 containing nucleotides 252-537 or 35-530 of SEQ ID NO:2, or a functional portion of SEQ ID NO:48 containing nucleotides 280-560 of SEQ ID NO:48, a composition having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, In some embodiments, compositions can be administered that contain a two-vector nucleic acid expression system, such as a system that utilizes two AAV vectors (e.g., an AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B vector) that collectively comprise a polynucleotide having at least one polynucleotide sequence (polynucleotide having at least one polynucleotide sequence identity of at least one of the polynucleotides) that have at least one polynucleotide sequence identity of at least one of the polynucleotides.

[0264] The two-vector system may be an overlapping dual vector system containing a first AAV vector and a second AAV vector. The overlapping dual vector system may include a first AAV vector including a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO: 3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 3), and a second AAV vector including a polynucleotide encoding a C-terminal portion of a stereocillin protein, where the 3' end of the stereocillin coding sequence of the first vector overlaps with the 5' end of the stereocillin coding sequence of the second vector. In another example, the two-vector system may be a trans-splicing dual vector system containing a first AAV vector and a second AAV vector. The trans-splicing dual vector system may include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3) and a second AAV vector that includes a splice acceptor signal sequence 5' of a polynucleotide encoding a C-terminal portion of a stereocillin protein. In another example, the two-vector system may be a dual hybrid vector system containing a first AAV vector and a second AAV vector.The dual hybrid vector system may include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3), a splice donor signal sequence 3' of the polynucleotide, and a first recombinogenic region 3' of the splice donor signal sequence, and a second AAV vector that includes a polynucleotide encoding a second recombinogenic region, a splice acceptor signal sequence 3' of the recombinogenic region, and a C-terminal portion of a stereocillin protein 3' of the splice acceptor signal sequence. In yet another example, the two-vector system can be a split intein trans-splicing system that includes a first AAV vector and a second AAV vector. A split intein trans-splicing two vector system can include a first AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding an N-terminal portion of a stereocillin protein (e.g., an N-terminal portion of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3) and a polynucleotide encoding an N-terminal intein (N-intein) 3' to such polynucleotide, and a second AAV vector that includes a STRC promoter operably linked to a polynucleotide encoding a C-terminal intein (C-intein) and a polynucleotide encoding a C-terminal portion of a stereocillin protein 3' to such polynucleotide.The above two-vector system may additionally include regulatory sequences, such as enhancers, poly(A) sequences, and STRC untranslated regions (UTRs, e.g., 5'UTR and / or 3'UTR), that are not part of the promoter described herein.

[0265] A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into a semicircular canal, e.g., the horizontal semicircular canal), to treat vestibular dysfunction.

[0266] After administering the composition to the patient, a practitioner of the art can monitor the expression of the therapeutic protein encoded by the transgene and the patient's improvement in response to treatment by a variety of methods.For example, a practitioner can monitor the patient's vestibular function after administering the composition by performing standard tests such as eye movement tests (e.g., electronystagmography (ENG) or video nystagmography (VNG)), tests of the vestibulo-ocular reflex (VOR) (e.g., head impulse test (Halmagyi-Curthoys test), which can be performed at the bedside or using video head impulse testing (VHIT), or caloric reflex test), stabilometry, rotary-chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), and balance tests in specialized clinics such as those described in Mancini et al., Eur J Phys Rehabil Med 46:239 (2010). A finding that the patient exhibits improved vestibular function in one or more of the tests after administration of the composition compared to the vestibular function test results before administration of the composition indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as necessary.

[0267] Exemplary embodiments of the present invention are described in the following enumerated paragraphs. E1. A polynucleotide comprising a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48, or a functional portion thereof comprising nucleotides 280-560 of SEQ ID NO:48, operably linked to a polynucleotide encoding a heterologous expression product.

[0268] E2. A polynucleotide comprising a STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2, operably linked to a polynucleotide encoding a heterologous expression product.

[0269] E3. The polynucleotide of E1, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48.

[0270] E4. The polynucleotide of E1 or E3, wherein said STRC promoter has the sequence of SEQ ID NO:48. E5. The polynucleotide of E1, wherein the functional portion of SEQ ID NO:48 comprises or consists of nucleotides 280 to 560 of SEQ ID NO:48.

[0271] E6. The polynucleotide of E1, wherein the functional portion of SEQ ID NO:48 comprises or consists of nucleotides 280 to 564 of SEQ ID NO:48. E7. The polynucleotide of E1, wherein the functional portion of SEQ ID NO:48 comprises or consists of nucleotides 124 to 560 of SEQ ID NO:48.

[0272] E8. The polynucleotide of E1, wherein the functional portion of SEQ ID NO:48 comprises or consists of nucleotides 124 to 564 of SEQ ID NO:48. E9. The polynucleotide of E1, wherein the functional portion of SEQ ID NO:48 comprises or consists of nucleotides 1 to 560 of SEQ ID NO:48.

[0273] E10. The polynucleotide of E2, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0274] E11. The polynucleotide of E2 or E10, wherein said STRC promoter consists of SEQ ID NO:1. E12. The polynucleotide of E2, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0275] E13. The polynucleotide of E2 or E12, wherein the functional portion of SEQ ID NO:2 comprises or consists of nucleotides 252 to 537 of SEQ ID NO:2. E14. The polynucleotide of E2 or E12, wherein the functional portion of SEQ ID NO:2 comprises or consists of nucleotides 120 to 537 of SEQ ID NO:2.

[0276] E15. The polynucleotide of E2 or E12, wherein the functional portion of SEQ ID NO:2 comprises or consists of nucleotides 35 to 530 of SEQ ID NO:2. E16. The polynucleotide of E2 or E12, wherein said STRC promoter consists of SEQ ID NO:2.

[0277] E17. The polynucleotide of any one of E1-E16, wherein the heterologous expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0278] E18. The proteins Actin Gamma 1 (ACTG1), Fascin Actin-Bundling Protein 2, Retinal (FSCN2), Radixin (RDX), POU Class 4 Homeobox 3 (POU4F3), TRIO and F-actin binding protein (TRIOBP), Taperin (TPRN), Xin Actin Binding Repeat Containing 2 (XIRP2), Atonal BHLH transcription factor 1 (ATOH1), and the transcriptional repressor Growth Factor Independent 1 (GFI1), Cholinergic Nicotinic Receptor Alpha 9 Subunit (CHRNA9), Cholinergic Nicotinic Receptor Alpha 10 Subunit (CHRNA10), Calcium and Integrin Binding Family Member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), MKRN2 Opposite Strand (MKRN2OS), LIM Homeobox Protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain-Containing Cysteine-Rich Protein 1 (Glutaredoxin Domain Containing Cysteine-Rich Protein 1 (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+ Transporting 2 (ATPase Plasma Membrane Ca2+ Transporting 2)2) (ATP2B2), calcium and integrin binding family member 2 (CIB2), voltage-dependent calcium channel auxiliary subunit alpha 2 delta 4 (CACNA2D4), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), peripherin 2 (PRPH2), solute carrier family 8 member A2 (SLC8A2), zinc finger CCHC-type containing protein 12 (ZCCHC12), leucine rich transmembrane and O-methyltransferase domain containing Containing (LRTOMT2, LRTOMT1), USH1 protein network component harmonin (USH1C), solute carrier family 26 member 5 (SLC26A5), Piezo-type mechanosensitive ion channel component 2 (PIEZO2), extracellular leucine rich repeat and fibronectin type III domain containing 1 (ELFN1), tetratricopeptide repeat protein 24 (TTC24), dystrotelin (DYTN), coiled-coil glutamic acid rich protein 2 (CCER2), leucine rich repeat and transmembrane domain containing protein 2 (LRTM2), voltage-gated potassium channel subfamily A member 10 (KCNA10), clarin 1 (CLRN1), clarin 2 (CLRN2), SKI family transcriptional corepressor 1 (SKI Family Transcriptional Core ... Corepressor 1) (SKOR1), Tctex1 domain-containing protein 1 (TCTEX1D1), Fc receptor-like B (FCRLB), glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2), serpin family E member 3 (SERPINE3), Nescient helix-loop-helix 1 (NescientThe polynucleotide of E17, which is Helix-loop Helix 1 (NHLH1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), activating transcription factor 6 (ATF6), eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), serine / threonine protein kinase / endoribonuclease IRE1 (IRE1), Whirlin (WHRN), oncomodulin (OCM), LIM homeobox 1 (Isl1), transmembrane and tetratricopeptide repeat containing 4 (TMTC4), binding immunoglobulin protein (BIP), or voltage-gated potassium channel subfamily Q member 4 (KCNQ4).

[0279] E19. A nucleic acid vector comprising the polynucleotide according to any one of E1 to E18. E20. A nucleic acid vector comprising a STRC promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48, or a functional portion thereof comprising nucleotides 280-560 of SEQ ID NO:48.

[0280] E21. A nucleic acid vector comprising a STRC promoter having (i) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or (ii) at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0281] E22. The nucleic acid vector of E20, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:48.

[0282] E23. The nucleic acid vector of E20 or E22, wherein said STRC promoter has the sequence of SEQ ID NO:48. E24. The nucleic acid vector of E20, wherein the functional part of SEQ ID NO:48 comprises or consists of nucleotides 280 to 560 of SEQ ID NO:48.

[0283] E25. The nucleic acid vector of E20, wherein the functional part of SEQ ID NO:48 comprises or consists of nucleotides 280 to 564 of SEQ ID NO:48. E26. The nucleic acid vector of E20, wherein the functional part of SEQ ID NO:48 comprises or consists of nucleotides 124 to 560 of SEQ ID NO:48.

[0284] E27. The nucleic acid vector of E20, wherein said functional part of SEQ ID NO:48 comprises or consists of nucleotides 124 to 564 of SEQ ID NO:48. E28. The nucleic acid vector of E20, wherein said functional part of SEQ ID NO:48 comprises or consists of nucleotides 1 to 560 of SEQ ID NO:48.

[0285] E29. The nucleic acid vector of E21, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0286] E30. The nucleic acid vector of E21 or E29, wherein said STRC promoter consists of SEQ ID NO:1. E31. The nucleic acid vector of E21, wherein the STRC promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2.

[0287] E32. The nucleic acid vector of E21 or E31, wherein said functional portion of SEQ ID NO:2 comprises or consists of nucleotides 252 to 537 of SEQ ID NO:2. E33. The nucleic acid vector of E21 or E31, wherein said functional portion of SEQ ID NO:2 comprises or consists of nucleotides 120 to 537 of SEQ ID NO:2.

[0288] E34. The nucleic acid vector of E21 or E31, wherein said functional portion of SEQ ID NO:2 comprises or consists of nucleotides 35 to 530 of SEQ ID NO:2. E35. The nucleic acid vector of E20 or E31, wherein said STRC promoter consists of SEQ ID NO:2.

[0289] E36. The nucleic acid vector of any one of E20 to E35, wherein said STRC promoter is operably linked to a polynucleotide encoding a heterologous expression product. E37. The nucleic acid vector of E36, wherein the heterologous expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0290] E38. The protein is selected from the group consisting of ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, EPS8, EPS8L2, ESPN, and ESP The nucleic acid vector of E37, which is NL, PRPH2, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, GRXCR2, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, Isl1, TMTC4, BIP, or KCNQ4.

[0291] E39. The nucleic acid vector according to any one of E20 to E35, wherein the STRC promoter is operably linked to a polynucleotide encoding the N-terminal portion of a stereocillin protein without encoding a full-length stereocillin protein.

[0292] E40. The nucleic acid vector of E39, wherein the nucleic acid vector is a first nucleic acid vector in a two-vector system and further comprises a second nucleic acid vector comprising a polynucleotide encoding a C-terminal portion of a stereocillin protein without encoding a full-length stereocillin protein.

[0293] E41. The nucleic acid vector according to any one of E19 to E40, wherein the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome. E42. The nucleic acid vector of E41, wherein the nucleic acid vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, and lentivirus.

[0294] E43. The nucleic acid vector of E42, wherein said viral vector is an AAV vector. E44. The nucleic acid vector of E43, wherein the AAV vector has an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid.

[0295] E45. A composition comprising the nucleic acid vector according to any one of E19 to E44. E46. The composition of E45, further comprising a pharma- ceutically acceptable carrier, diluent, or excipient.

[0296] E47. A cell comprising a polynucleotide according to any one of E1 to E18 or a nucleic acid vector according to any one of E19 to E44. E48. The cell of E47, wherein said cell is a hair cell.

[0297] E49. The cell of E48, wherein said hair cell is a mammalian hair cell. E50. The cell of E49, wherein said mammalian hair cell is a human hair cell. E51. The cell according to any one of E48 to E50, wherein the hair cell is a cochlear hair cell.

[0298] E52. The cell of E51, wherein said cochlear hair cell is an outer hair cell. E53. The cell of E51, wherein said cochlear hair cell is an inner hair cell. E54. The cell according to any one of E48 to E50, wherein the hair cell is a vestibular hair cell.

[0299] E55. The cell of E54, wherein the vestibular hair cell is a type II vestibular hair cell. E56. The cell of E54, wherein the vestibular hair cell is a type I vestibular hair cell. E57. A method for expressing a heterologous expression product in a hair cell, comprising contacting said hair cell with a nucleic acid vector described in any one of E19 to E44 or a composition described in E45 or E46.

[0300] E58. The method of E57, wherein said expression product is specifically expressed in hair cells. E59. A method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss, auditory neuropathy, or hearing loss), comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector described in any one of E19-E44 or a composition described in E45 or E46.

[0301] E60. A method of treating a subject having or at risk of developing tinnitus, comprising administering to the inner ear of said subje...

Claims

1. A polynucleotide comprising an STRC promoter operably linked to a polynucleotide encoding a heterologous expression product, wherein the STRC promoter has (i) at least 85% sequence identity to SEQ ID NO:48 or a functional portion thereof comprising nucleotides 280 to 560 of SEQ ID NO:48, (ii) at least 85% sequence identity to SEQ ID NO:1, or (iii) at least 85% sequence identity to SEQ ID NO:2 or a functional portion thereof comprising nucleotides 252 to 537 or 35 to 530 of SEQ ID NO:

2.

2. The polynucleotide of claim 1, wherein the STRC promoter has the sequence of SEQ ID NO:48, SEQ ID NO:1, or SEQ ID NO:

2. (a) the functional portion of SEQ ID NO:48 comprises nucleotides 280 to 560 of SEQ ID NO:48, nucleotides 280 to 564 of SEQ ID NO:48, nucleotides 124 to 560 of SEQ ID NO:48, nucleotides 124 to 564 of SEQ ID NO:48, or nucleotides 1 to 560 of SEQ ID NO:48; or (b) the polynucleotide of claim 1, wherein the functional portion of SEQ ID NO:2 comprises nucleotides 252 to 537 of SEQ ID NO:2, nucleotides 120 to 537 of SEQ ID NO:2, or nucleotides 35 to 530 of SEQ ID NO:

2.

4. 2. The polynucleotide of claim 1, wherein the heterologous expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system, or a microRNA.

5. A nucleic acid vector comprising the polynucleotide of claim 1.

6. a) a first nucleic acid vector comprising an STRC promoter operably linked to a first polynucleotide encoding an N-terminal portion of a stereocillin protein, said STRC promoter having (i) at least 85% sequence identity to SEQ ID NO:48 or a functional portion thereof comprising nucleotides 280-560 of SEQ ID NO:48, (ii) at least 85% sequence identity to SEQ ID NO:1, or (iii) at least 85% sequence identity to SEQ ID NO:2 or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2; b) a second nucleic acid vector comprising a second polynucleotide encoding the C-terminal portion of the stereocillin protein.

7. 7. The two-vector system of claim 6, wherein the STRC promoter has the sequence of SEQ ID NO:48, SEQ ID NO:1, or SEQ ID NO:

2. (a) the functional portion of SEQ ID NO:48 comprises nucleotides 280 to 560 of SEQ ID NO:48, nucleotides 280 to 564 of SEQ ID NO:48, nucleotides 124 to 560 of SEQ ID NO:48, nucleotides 124 to 564 of SEQ ID NO:48, or nucleotides 1 to 560 of SEQ ID NO:48; or (b) the functional portion of SEQ ID NO:2 comprises nucleotides 252 to 537 of SEQ ID NO:2, nucleotides 120 to 537 of SEQ ID NO:2, or nucleotides 35 to 530 of SEQ ID NO:

2.

9. 7. The two-vector system of claim 6, wherein the first polynucleotide partially overlaps with the second polynucleotide, and when introduced into a mammalian cell, the first nucleic acid vector and the second nucleic acid vector undergo homologous recombination to form a recombinant polynucleotide encoding a full-length stereocillin protein.

10. the first nucleic acid vector comprises a splice donor signal sequence located 3' to the first polynucleotide; the second nucleic acid vector comprises a splice acceptor signal sequence located 5' to the second polynucleotide; the first nucleic acid vector comprises a splice donor signal sequence located 3' to the first polynucleotide and a first recombinogenic region located 3' to the splice donor signal sequence; 7. The two-vector system of claim 6, wherein the second nucleic acid vector comprises a second recombinogenic region and a splice acceptor signal sequence located 3' of the recombinogenic region and 5' of the second polynucleotide.

11. 7. The two-vector system of claim 6, wherein the first polynucleotide and the second polynucleotide do not overlap.

12. 7. The two-vector system of claim 6, wherein the second nucleic acid vector further comprises a STRC promoter operably linked to the second polynucleotide, wherein the STRC promoter has (i) at least 85% sequence identity to SEQ ID NO:48 or a functional portion thereof comprising nucleotides 280-560 of SEQ ID NO:48, (ii) at least 85% sequence identity to SEQ ID NO:1, or (iii) SEQ ID NO:2 or a functional portion thereof comprising nucleotides 252-537 or 35-530 of SEQ ID NO:2, and wherein the STRC promoter is located 5' to the second polynucleotide.

13. 7. The two-vector system of claim 6, wherein the first nucleic acid vector further comprises a polynucleotide encoding an N-terminal intein (N-intein) located 3' to the first polynucleotide, and the second nucleic acid vector further comprises a polynucleotide encoding a C-terminal intein (C-intein) located between the STRC promoter and the second polynucleotide.

14. The two-vector system of claim 6, wherein the first polynucleotide encoding the N-terminal portion of the stereocillin protein and the second polynucleotide encoding the C-terminal portion of the stereocillin protein do not contain introns.

15. A method for expressing a heterologous expression product in a hair cell, the method comprising contacting the hair cell with the nucleic acid vector of claim 5.

16. 10. A method for expressing a stereocillin protein in a human hair cell, the method comprising contacting the human hair cell with the two-vector system of claim 6.

17. 6. The nucleic acid vector of claim 5 for use in a method for treating a subject having or at risk of developing sensorineural hearing loss or vestibular dysfunction.

18. 6. The nucleic acid vector of claim 5 for use in a method for treating a subject having or at risk of developing tinnitus.

19. The nucleic acid vector of claim 5 for use in a method for inducing or increasing hair cell regeneration, inducing or increasing hair cell maturation, preventing or reducing hair cell damage or cell death, increasing hair cell survival, or improving hair cell function in a subject in need thereof.

20. 10. The two-vector system of claim 6 for use in a method for increasing expression of STRC in a subject in need thereof.

21. 20. The nucleic acid vector of claim 19, wherein the subject has or is at risk of developing hearing loss or vestibular dysfunction.

22. The object is (a) has or is identified as having a STRC mutation; (b) the nucleic acid vector of claim 17, having hearing loss, autosomal recessive 16 (DFNB16).

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