Recombinant adeno associated virus and uses thereof

Abstract

Provided herein are polynucleotides comprising an engineered 3' untranslated region (3' UTR) and methods for using the same to treat hearing loss (e.g., genetic hearing loss) Also provided are vectors, recombinant adeno-associated virus (rAAV) compositions, cells, and compositions comprising the polynucleotides.

Description

RECOMBINANT ADENO ASSOCIATED VIRUS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to United States Provisional Application Serial Number 63 / 555,605, filed February 20, 2024, the contents of which are hereby incorporated by reference herein in their entirety.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a sequence listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said ST.26 copy, created on February 18, 2025, is named “216253_seqlist.xml” and is 81,499 bytes in size.BACKGROUND

[0003] Congenital hearing loss affects about 1 in 500 newborns and most of the cases are genetic in cause. For example. SLC26A4 is among the top four genes that are associated with hearing loss, and mutations in the gene can cause Pendred syndrome (PDS) and autosomal recessive non-syndromic deafness 4 (DFNB4). SLC26A4 encodes the protein pendrin, which promotes transmembrane exchange of anions (such as CF and I’) and bases (such as HCO3' and OH') across plasma membranes of epithelial cells. In the mouse inner ear, pendrin is expressed in mitochondria-rich cells of the endolymphatic sac, transitional cells of the vestibule, outer sulcus cells, root cells and spindle cells in the cochlea. Pendrin expression in these cells is required to keep the ion balance in the inner ear and is thus necessary7for hearing.

[0004] Replacement gene therapy provides a unique opportunity to treat diseases and disorders associated with the inner ear, such as genetic hearing loss. Expression of a functional copy of a mutated gene that is active in inner ear function (e.g, SLC26A4) can treat genetic hearing loss. However, promiscuous expression of the gene in cell types where the gene is not normally expressed can lead to harmful side effects. Accordingly, there is a need in the art for improved gene therapy compositions and methods that can efficiently and safely restore hearing in subjects impacted by genetic hearing loss.SUMMARY

[0005] Provided herein are polynucleotides comprising a coding sequence (e.g, a pendrin protein coding sequence) and an engineered 3' untranslated region (3' UTR) and methods for using the same to treat hearing loss (e.g, hearing loss associated with enlarged vestibularaqueduct, genetic hearing loss, Pendred syndrome, autosomal recessive nonsyndromic deafness 4). Also provided are vectors, recombinant adeno-associated virus (rAAV) compositions, cells, and compositions comprising the polynucleotides. The polynucleotides provided herein are particularly advantageous in that they can promote highly specific expression of the coding sequence in particular inner ear cells and thereby facilitate treatment of inner-ear associated diseases.

[0006] Accordingly, in one aspect, the present disclosure provides a polynucleotide comprising from 5' to 3': a) a pendrin protein coding sequence; and b) an engineered 3' UTR of 15 to about 2,000 nucleotides in length, the engineered 3' UTR comprising two or more non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11. In some embodiments, the pendrin protein is a human pendrin protein. In some embodiments, the pendrin protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1. In some embodiments, the pendrin protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the pendrin protein coding sequence comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In some embodiments, the pendrin protein coding sequence comprises the nucleotide sequence of SEQ ID NO: 2.

[0007] In some embodiments, the engineered 3' UTR comprises two, three, four, five, six, seven, eight, or nine non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11. In some embodiments, the engineered 3' UTR comprises, from 5' to 3': a nucleotide sequence that is at least 80% identical to SEQ ID NO: 3; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 4; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 8; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 9; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the engineered 3' UTR comprises, from 5' to 3': anucleotide sequence consisting of SEQ ID NO: 3; a nucleotide sequence consisting of SEQ ID NO: 4; a nucleotide sequence consisting of SEQ ID NO: 5; a nucleotide sequence consisting of SEQ ID NO: 6; a nucleotide sequence consisting of SEQ ID NO: 7; a nucleotide sequence consisting of SEQ ID NO: 8; a nucleotide sequence consisting of SEQ ID NO: 9; a nucleotide sequence consisting of SEQ ID NO: 10; and a nucleotide sequence consisting of SEQ ID NO: 11.

[0008] In some embodiments, the engineered 3' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the engineered 3' UTRcomprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 13.

[0009] In some embodiments, the polynucleotide further comprises a 5' untranslated region (5' UTR) positioned 5' of the pendrin protein coding sequence. In some embodiments, the 5' UTR is 15 to about 200 nucleotides in length. In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 17. In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the 5' UTR consists of the nucleotide sequence of SEQ ID NO: 17.

[0010] In some embodiments, the polynucleotide further comprises a promoter operably linked to the pendrin protein coding sequence. In some embodiments, the promoter is a chicken betaactin (CBA) promoter, a human SLC26A4 promoter, a CAG promoter, a cytomegalovirus (CMV) promoter, a eukaryotic translation elongation factor la (EFla) promoter, a T7 polymerase promoter, or a simian vacuolating virus 40 (SV40) promoter. In some embodiments, the CBA promoter comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 18 or 19. In some embodiments, the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 18 or 19. In some embodiments, the CBA promoter consists of the nucleotide sequence of SEQ ID NO: 18 or 19.

[0011] In some embodiments, the polynucleotide further comprises an intron element positioned between the promoter and the pendrin protein coding sequence. In some embodiments, the intron element is an SV-40 intron element or a CBA intron element. In some embodiments, the intron element comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 27. In some embodiments, the intron element comprises the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the intron element consists of the nucleotide sequence of SEQ ID NO: 27.

[0012] In some embodiments, the polynucleotide further comprises an enhancer positioned 5' of the promoter. In some embodiments, the enhancer comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 28. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the enhancer consists of the nucleotide sequence of SEQ ID NO: 28.

[0013] In some embodiments, the polynucleotide further comprises a polyadenylation sequence positioned 3' of the engineered 3' UTR. In some embodiments, the polyadenylation sequence is a bovine grow th hormone polyadenylation sequence. In some embodiments, thepolyadenylation sequence comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 29. In some embodiments, the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the polyadenylation sequence consists of the nucleotide sequence of SEQ ID NO: 29.

[0014] In some embodiments, the polynucleotide is capable of expressing pendrin in cells that normally express the SLC26A4 gene. In some embodiments, the polynucleotide is capable of expressing pendrin in inner ear cells. In some embodiments, the polynucleotide is capable of expressing pendrin in root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.

[0015] In some embodiments, the polynucleotide further comprises an inverted terminal repeat (ITR) positioned at the 5' end (5' ITR) and / or an ITR positioned at the 3' end (3' ITR). In some embodiments, the ITR is derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. In some embodiments, the ITR is derived from AAV2. In some embodiments, the polynucleotide comprises: a 5' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 32; and / or a 3' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 33. In some embodiments, the polynucleotide comprises: a 5' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 32; and a 3' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 33. In some embodiments, the 5' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the 3' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 33.

[0016] In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 34, 37, 41, and 42. In some embodiments, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 34, 37, 41, and 42. In some embodiments, the polynucleotide consists of the nucleotide sequence of any one of SEQ ID NOs: 34, 37, 41, and 42.

[0017] In another aspect, provided herein is a polynucleotide comprising an engineered 3' untranslated region (3' UTR) of 15 to about 2,000 nucleotides in length, the engineered 3' UTR comprising two or more non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11. In some embodiments, the engineered 3' UTR comprises three, four, five, six, seven, eight, or nine non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11. In some embodiments, the engineered 3' UTR comprises, from 5' to 3': a nucleotide sequence that is at least 80% identical to SEQ IDNO: 3; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 4; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 8; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 9; a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the engineered 3' UTR comprises, from 5' to 3': a nucleotide sequence consisting of SEQ ID NO: 3; a nucleotide sequence consisting of SEQ ID NO: 4; a nucleotide sequence consisting of SEQ ID NO: 5; a nucleotide sequence consisting of SEQ ID NO: 6; a nucleotide sequence consisting of SEQ ID NO: 7; a nucleotide sequence consisting of SEQ ID NO: 8; a nucleotide sequence consisting of SEQ ID NO: 9; a nucleotide sequence consisting of SEQ ID NO: 10; and a nucleotide sequence consisting of SEQ ID NO: 11.

[0018] In some embodiments, the engineered 3' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the engineered 3' UTR comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 13.

[0019] In some embodiments, the polynucleotide further comprises a transgene positioned 5' of the engineered 3' UTR.

[0020] In some embodiments, the polynucleotide further comprises a 5' untranslated region (5' UTR) positioned 5' of the transgene. In some embodiments, the 5' UTR is 15 to about 200 nucleotides in length. In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 17. In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the 5' UTR consists of the nucleotide sequence of SEQ ID NO: 17.

[0021] In some embodiments, the polynucleotide further comprises a promoter operably linked to the transgene.

[0022] In some embodiments, the polynucleotide is capable of expressing the transgene in inner ear cells. In some embodiments, the polynucleotide is capable of expressing the transgene in root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.

[0023] In some embodiments, the polynucleotide further comprises an inverted terminal repeat (ITR) positioned at the 5' end (5' ITR) and / or an ITR positioned at the 3' end (3' ITR).

[0024] In another aspect, the present disclosure provides a vector comprising a polynucleotide described herein. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the viral vector is an AAV vector.

[0025] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising: a capsid protein; and a polynucleotide described herein. In some embodiments, the rAAV has tropism for a subset of inner ear cells that normally express the SLC26A4 gene. In some embodiments, the rAAV has tropism for cells of the cochlea. In some embodiments, the capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV-PHP.B capsid protein, an AAV-S capsid protein, or a variant thereof. In some embodiments, the capsid protein is an AAV-S capsid protein.

[0026] In another aspect, the present disclosure provides a cell comprising a polynucleotide, vector, or rAAV described herein.

[0027] In another aspect, the present disclosure provides a composition comprising a polynucleotide, vector, rAAV, or cell described herein and a pharmaceutically acceptable excipient.

[0028] In another aspect, the present disclosure provides a method for specifically expressing pendrin in cells that normally express the SLC26A4 gene in a subject, the method comprising administering to the subject an effective amount of a polynucleotide, vector, rAAV, cell, or composition described herein.

[0029] In another aspect, the present disclosure provides a method for treating hearing loss in a subject in need thereof, the method comprising administering to the subject an effective amount of a polynucleotide, vector, rAAV, cell, or composition described herein. In some embodiments, the hearing loss is genetic hearing loss. In some embodiments, the subject has or is suspected to have an enlarged vestibular aqueduct (EVA). In some embodiments, the hearing loss is associated with a mutation in the SLC26A4 gene. In some embodiments, the subject has or is suspected to have Pendred syndrome (PDS) and / or autosomal recessive nonsyndromic deafness 4 (DFNB4).

[0030] In another aspect, the present disclosure provides a method for treating a pendrin- associated disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a polynucleotide, vector, rAAV, cell, or composition described herein. In some embodiments, the subject has or is suspected to have an enlarged vestibularaqueduct (EVA). In some embodiments, the pendrin-associated disease is Pendred syndrome (PDS) or autosomal recessive nonsyndromic deafness 4 (DFNB4).

[0031] In another aspect, the present disclosure provides a method for specifically expressing a transgene in inner ear cells in a subject, the method comprising administering to the subject an effective amount of a polynucleotide, vector, rAAV, cell, or composition described herein.

[0032] In some embodiments of the methods provided herein, the subj ect is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human mammal. In some embodiments, the non-human mammal is a mouse, a rat, or a non-human primate.

[0033] In some embodiments of the methods provided herein, the step of administering results in expression of pendrin protein in inner ear cells. In some embodiments, the inner ear cells are root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells. In some embodiments, the administration is via injection. In some embodiments, the injection is through the round window membrane of the cochlea, into the scala media of the cochlea, into the scala vestibuli of the cochlea, into a semicircular canal of the inner ear. or into the saccule or the utricle of the inner ear.

[0034] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of certain embodiments and also from the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

[0036] FIG. 1 shows a schematic representation of a pendrin activity assay, according to certain aspects of the present disclosure. HEK293 Phoenix Eco cells were transduced with AAV and then transfected with a plasmid encoding an anion-sensitive YFP mutant. 3 days post-transduction, pendrin anion exchange activity was measured by YFP fluorescence quenching.

[0037] FIG. 2 shows schematic representation of exemplary AAV vectors, according to certain aspects of the present disclosure. CBA: chicken beta-actin; h. Pendrin 5'UTR: humanSLC26A4 5' untranslated region; h. Pendrin CDS: human pendrin protein coding sequence; h. Pendrin 3'UTR: human SLC26A4 3' untranslated region; bGH polyA: bovine growth hormone polyadenylation signal; HA: human influenza hemagglutinin tag; ITR: inverted terminal repeat.

[0038] FIG. 3 shows the results of a pendrin activity7assay based on YFP fluorescence quenching in cells transduced with an AAV vector, according to certain aspects of the present disclosure. The top panel shows that rAAVs with the SLC26A4-UTR vector produce a functional pendrin protein capable of anion exchange in a dose-dependent manner, according to certain aspects of the present disclosure. YFP fluorescence quenching was measured in cells transduced with the indicated MOIs of AAV or formulation buffer (FB). Data are mean ± standard deviation (n = 3) and are plotted as the % of the starting fluorescence of each well in a high Cl’ solution prior to the addition of the high I’ solution (set as 100%). Statistical analyses were performed using one-way ANOVA with Tukey post-hoc analysis. ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001). The bottom panel shows that pendrin expression correlates with activity, according to certain aspects of the present disclosure. After measuring pendrin activity7, expression of pendrin was detected by immunofluorescence with an anti-pendrin antibody.

[0039] FIG. 4 shows that quenching of YFP fluorescence by pendrin-mediated anion exchange is reversible, according to certain aspects of the present disclosure. YFP fluorescence quenching was measured in cells transduced with the indicated MOIs of AAV or formulation buffer (FB). Data are mean ± standard deviation (ra = 3) and are plotted as the % of the starting fluorescence of each well in a high Cl’ solution prior to the addition of the high T solution (set as 100%). After reading YFP fluorescence, cells were returned to growth media (media change) and allowed to incubate for 2 min at room temperature prior to running the pendrin activity assay7again as described.

[0040] FIG. 5 shows that SLC26A4-UTR AAVs injected at Pl-2 preserved ABR threshold in the SLC26A4 L236P mouse model, according to certain aspects of the present disclosure. AAV was injected through RWM into the cochleae of the SLC26A4 L236P homozygous mice (HOM) at varying doses: low dose (n=31), mid dose (n=60), high dose (n=91), and higher dose (n=60), and ABR was recorded around P30. Uninjected L236P HOM mice (n=129) were used to compare to injected groups. The ABR thresholds in the mid, high, and higher dose groups were significantly different from the ABR thresholds in the uninjected group (****, p<0.0001, Two-way ANOVA Dunnetf s adjusted), while the thresholds in the low dose group were notsignificantly different from those of the uninjected group (P > 0.05, Two-way ANOVA Dunnett’s adjusted). ABR thresholds from CBA / CaJ wild type (WT) mice without SLC26A4 L236P mutations and with normal hearing thresholds (n=l 0) are also shown. All data show n represent median ABR threshold values ± 95% confidence interval (CI) at the indicated frequencies.DETAILED DESCRIPTION

[0041] Provided herein are polynucleotides comprising a coding sequence (e.g., a pendrin protein coding sequence) and an engineered 3' untranslated region (3' UTR), and methods for using the same to treat hearing loss (e.g., hearing loss associated with enlarged vestibular aqueduct, genetic hearing loss, Pendred syndrome, autosomal recessive nonsyndromic deafness 4). Also provided are vectors, recombinant adeno-associated virus (rAAV) compositions, cells, and compositions comprising the polynucleotides.

[0042] SLC26A4 is among the top four genes that are associated with hearing loss (Sloan- Heggen et al., 2016, Hum. Genet. 135:441-450). Mutations in the SLC26A4 gene can cause genetic hearing loss associated with, e.g. , Pendred syndrome (PDS) and autosomal recessive non-syndromic deafness 4 (DFNB4). SLC26A4 encodes the protein pendrin. which promotes transmembrane exchange of anions (such as Cl" and I") and bases (such as HCO3" and OH") across plasma membranes of epithelial cells (Alper and Sharma, 2013, Mol. Aspects Med. 34:494-515). In the mouse inner ear, pendrin is expressed in mitochondria-rich cells of the endolymphatic sac, transitional cells of the vestibule, outer sulcus cells, root cells and spindle cells in the cochlea (Everett et al., 1999, Proc. Nat. Acad. Sci. 96:9727-9732; Everett et al., 2001, Hum. Mol. Genet. 10: 153-161; Rovaux et al., 2003, Journal of the Association for Research in Otolaryngology 4:394-404; Li et al., 2013, PLoS Genet. 9:el003641). Pendrin expression in these cells is required to keep the ion balance in the inner ear and is thus necessary for bearing (Li et al.. 2013, PLoS Genet. 9:el003641; Wangemann etal., 2006. Am. J. Physio. Renal Physiol. 292:F1345-F1353).

[0043] The present disclosure is based, at least in part, on the surprising discovery by the inventors that inclusion of an engineered 3' UTR comprising two or more sequences from the SLC26A4 gene 3' UTR in a polynucleotide with a coding sequence (e.g., as part of an rAAV vector provided herein) can promote tissue- and cell type-specific expression of the coding sequence (e.g., when administered to a subject). As demonstrated in the Examples herein, the expression pattern is specific to cells that endogenously express the SLC26A4 gene. Furthermore, the Examples herein demonstrate that an engineered 3' UTR as described hereincan be incorporated into an rAAV and used to preserve hearing in subjects with hearing loss (e.g, mouse models of DFNB4).I. Definitions

[0044] As used herein, the term “pendrin” refers to the anion exchange protein that in humans is encoded by the solute carrier family 26 member 4 (SLC26A4) gene. An exemplary' amino acid sequence of a pendrin polypeptide is provided as SEQ ID NO: 1.

[0045] As used herein, the term “engineered” in the context of an engineered nucleotide sequence (e.g , an engineered 3' UTR) indicates that the nucleotide sequence contains one or more sequence alterations (e.g., insertions, deletions, and / or substitutions) relative to a naturally occurring nucleotide sequence.

[0046] As used herein in the context of comparing nucleic acid sequences or amino acid sequences, the term “percent identity” refers to the percentage of nucleic acid or amino acid residues of a candidate sequence that are identical to the nucleic acid or amino acid residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill of one in the art, for instance, using publicly available computer software, such as BLAST, BLAST-2, BLAST-P, BLAST- N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL. or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0047] As used herein, the term “transgene” refers to a non- AAV nucleic acid sequence that encodes a polypeptide or non-coding RNA (e.g., a miRNA, shRNA. siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA apta / me. or RNA aptamer).

[0048] As used herein, the term “coding sequence” refers to the portion of a complementary DNA (cDNA) or mRNA that encodes a polypeptide, starting at the start codon and ending at the stop codon. A gene may have one or more coding sequences due to alternative splicing and / or alternative translation initiation. A coding sequence can be wild-type or engineered (e.g, to optimize protein expression from the coding sequence, or to reduce immunogenicity of the coding sequence).

[0049] As used herein, the term “gene regulatory' element” or “GRE” refers to a cv.s-acting nucleotide sequence, e.g, a DNA sequence, that regulates (e.g., controls, increases, or reduces) expression of an operably linked nucleotide sequence (e.g, transcription of a transgene). A GRE generally relies on one or more trans-acting molecules, such as transcription factors, to regulate gene expression. Thus, one GRE may regulate expression in different ways when it is in contact with different trans-acting molecules, for example, when it is in different types of cells. A GRE may comprise, e.g., one or more promoter elements and / or enhancer elements. A skilled artisan would appreciate that the promoter and enhancer elements in a gene may be close in location, and the term “promoter” may refer to a sequence comprising a promoter element and an enhancer element. Thus, the term “promoter” does not exclude an enhancer element in the sequence. The promoter and enhancer elements do not need to be derived from the same gene or species, and the sequence of each promoter or enhancer element may be either identical or substantially identical to the corresponding endogenous sequence in the genome. A promoter may be, but is not limited to, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter.

[0050] As used herein, the term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the transcription of a gene.

[0051] As used herein, the term “enhancer” refers to a DNA sequence that is capable of interacting with site-specific transcription factors to regulate gene expression in a cell-type specific manner. Enhancers may be located more distal to the transcription start site relative to a promoter. In some embodiments, enhancers confer cell-specific gene expression regulation by binding to the collection of transcription factors in a cell, which leads to transcriptional activation or inhibition through various mechanisms, e.g, recruitment of epigenetic enzymes that catalyze post-translational histone modifications, and recruitment of cofactors that promote DNA looping. Enhancers can be identified in the vicinity of the gene they regulate, or at a distance of hundreds of kilobases from their target genes. Multiple enhancers can act additively and redundantly to regulate gene expression.

[0052] As used herein, the term “intron element” refers to a nucleotide sequence, for example, a DNA sequence, that regulates (e.g., controls, increases, or reduces) expression of a transgene. In some embodiments, an intron element is a modified intron, e.g., a synthetic intron sequence. In some embodiments, an intron element is an exogenous intron element and is derived from an intron exogenous to the transgene it may regulate. In some embodiments, an intron element comprises a modified splice acceptor and / or splice donor resulting in more robust splicingactivity. While not wishing to be bound by theory, it is hypothesized that introns can increase transgene expression, for example, by reducing transcriptional silencing and enhancing mRNA export from the nucleus to the cytoplasm. A skilled worker will appreciate that synthetic intron sequences can be designed to mediate RNA splicing by introducing any consensus splicing motifs known in the art (e.g., in Sibley et al. (2016) Nature Reviews Genetics, 17, 407-21, which is incorporated by reference herein in its entirety). Exemplary intron sequences are provided in Lu etal. (2013) Molecular Therapy 21(5): 954-63, and / .u er al. (2017) Hum. Gene Ther. 28(1): 125-34, which are incorporated by reference herein in their entirety.

[0053] As used herein, the term “operably linked” is used to describe the connection between a gene regulatory' element (e.g., a promoter) and a coding sequence to be transcribed. The coding sequence is “operably linked” to the regulatory nucleotide sequence if the transcription of the coding sequence is controlled or influenced by the regulatory' nucleotide sequence. The regulatory nucleotide sequence may be in any orientation and / or at any' distance from the coding sequence, as long as the desired transcriptional activity is obtained. In some embodiments, the regulatory nucleotide sequence is upstream from the coding sequence.

[0054] As used herein, the term “posttranscriptional response element” refers to a nucleic acid sequence that, when transcribed, enhances expression of a gene (e.g., by adopting a particular tertiary' structure). Examples of posttranscriptional regulatory elements include, but are not limited to, woodchuck hepatitis virus posttranscriptional regulatory' element (WPRE), mouse RNA transport element (RTE), constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MP MV), and the 5' untranslated region of the human heat shock protein 70 (Hsp70 5' UTR).

[0055] As used herein, the term “polyadenylation sequence” refers to a DNA sequence that when transcribed into RNA constitutes a polyadenylation signal sequence. The polyadenylation sequence can be native (e.g., from the human SLC26A4 gene) or exogenous. The exogenous polyadenylation sequence can be a mammalian or a viral polyadenylation sequence (e.g., a bovine growth hormone polyadenylation sequence).

[0056] As used herein, the term “recombinant AAV” (rAAV) refers to an AAV that has been produced using recombinant methods. An “rAAV vector” is typically composed of. at a minimum, a coding sequence (e.g., a pendrin protein coding sequence) and 5' and 3' AAV inverted terminal repeats (ITRs) and may also comprise, for example, 5' and 3' untranslated regions (UTRs) and / or an expression control sequence (e.g., a polyadenylation sequence). Recombinant AAVs (rAAVs) may have tissue-specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s).The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.

[0057] As used herein, the term “vector” includes any construct, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid is capable of being transcribed.

[0058] As used herein, the term “pharmaceutically acceptable excipient” includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and does not cause disruptive physiological reactions, such as an unintended immune reaction. Pharmaceutically acceptable excipient include water, phosphate buffered saline, emulsions such as oil / water emulsion, and wetting agents. Compositions comprising such excipients are formulated by well-known conventional methods such as those set forth in Remington ’s Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy ” 20th edition. Lippincott, Williams. & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et cd., 3rd ed. Amer. Pharmaceutical Assoc.

[0059] As used herein, the term “subject” includes any human or non-human animal. In some embodiments, the subject is a human.

[0060] As used herein, the term “effective amount” refers to the amount of a substance that is sufficient to produce a desired effect. In the context of administration of a therapy (e.g., a polynucleotide, rAAV. or composition as described herein) to a subject, the term refers to the amount of the therapy that achieves a desired prophylactic or therapeutic effect in the subject.

[0061] As used herein, the terms “about” or “approximately,” when referring to a measurable value, encompasses variations of ±10% of a given value or range.II. Polynucleotides and vectors

[0062] In some aspects, provided herein are polynucleotides comprising an engineered 3' UTR useful for driving cell-type specific expression of a transgene (e.g., a protein coding sequence). In some embodiments, the polynucleotides further comprise a transgene. In some embodiments, the transgene comprises a protein coding sequence (e.g, a pendrin protein coding sequence).A. Engineered 3' UTR

[0063] Various embodiments of the polynucleotides provided herein comprise an engineered 3' UTR. In some embodiments, the engineered 3' UTR has a length of about 5 to about 2.500 nucleotides (e g., about 5 to about 2,000, about 5 to about 1,500, about 5 to about 1,000, about 10 to about 2,500, about 10 to about 2,000, about 10 to about 1,500, about 15 to about 2,500, about 15 to about 2,000, about 15 to about 1,500. about 25 to about 2,500, about 25 to about 2,000, about 25 to about 1,500, about 50 to about 2,500, about 50 to about 2,000, about 50 to about 1,500, about 100 to about 2,500, about 100 to about 2,000, about 100 to about 1,500, about 200 to about 2,500, about 200 to about 2,000, about 200 to about 1,500, about 300 to about 2,500, about 300 to about 2,000, or about 300 to about 1,500 nucleotides). In some embodiments, the engineered 3' UTR is about 15 to about 2,000 nucleotides in length.

[0064] In some embodiments, the engineered 3' UTR has a length of 5 to 2,500 nucleotides (e.g, 5 to 2,000, 5 to 1,500, 5 to 1,000, 10 to 2,500, 10 to 2,000, 10 to 1,500, 15 to 2,500, 15 to 2,000, 15 to 1,500, 25 to 2,500, 25 to 2,000, 25 to 1,500, 50 to 2,500, 50 to 2,000, 50 to 1,500, 100 to 2,500, 100 to 2,000, 100 to 1,500, 200 to 2,500, 200 to 2,000, 200 to 1,500, 300 to 2,500, 300 to 2,000. or 300 to 1,500 nucleotides). In some embodiments, the engineered 3' UTR is 15 to 2,000 nucleotides in length.

[0065] In some embodiments, the engineered 3' UTR has a length of less than about 2,500 nucleotides (e.g., less than about 2,400, less than about 2,300, less than about 2,200, less than about 2,100, less than about 2,000. less than about 1,900, less than about 1,800, less than about 1,700, less than about 1,600, less than about 1,500, less than about 1,400, less than about 1,300, less than about 1,200, less than about 1,100, less than about 1,100, less than about 950, less than about 900, less than about 850, less than about 800, less than about 750, less than about 700, less than about 650, less than about 600, less than about 550, or less than about 500 nucleotides). In some embodiments, the engineered 3' UTR is less than about 2,000 nucleotides in length.

[0066] In some embodiments, the engineered 3' UTR has a length of less than 2,500 nucleotides (e.g., less than 2,400, less than 2,300, less than 2,200. less than 2,100, less than 2,000, less than 1,900, less than 1,800, less than 1,700, less than 1,600, less than 1,500, less than 1,400, less than 1,300, less than 1,200, less than 1,100, less than 1,100, less than 950, less than 900, less than 850, less than 800, less than 750, less than 700, less than 650, less than 600, less than 550, or less than 500 nucleotides). In some embodiments, the engineered 3' UTR is less than 2.000 nucleotides in length.

[0067] In some embodiments, the engineered 3' UTR has a length of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65. about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140. about 150, about 160, about 170, about 180, about 190, about 200, about 220, about 240, about 260, about 280, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2.100. about 2,200, about 2,300, about 2.400. or about 2.500 nucleotides. In some embodiments, the engineered 3' UTR is about 400 nucleotides in length. In some embodiments, the engineered 3' UTR is about 375 nucleotides in length. In some embodiments, the engineered 3' UTR is about 370 nucleotides in length. In some embodiments, the engineered 3' UTR is about 366 nucleotides in length. In some embodiments, the engineered 3' UTR is about 365 nucleotides in length. In some embodiments, the engineered 3' UTR is about 360 nucleotides in length. In some embodiments, the engineered 3' UTR is about 355 nucleotides in length. In some embodiments, the engineered 3' UTR is about 350 nucleotides in length.

[0068] In some embodiments, the engineered 3' UTR comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a native sequence (e.g., a native sequence from the human genome).

[0069] In some embodiments, the 3' UTR comprises anucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a nucleotide sequence set forth in SEQ ID NO: 12.

[0070] In some embodiments, the engineered 3' UTR comprises two or more nucleotide sequences (e.g, three or more nucleotide sequences, four or more nucleotide sequences, five or more nucleotide sequences, six or more nucleotide sequences, seven or more nucleotide sequences, eight or more nucleotide sequences, nine or more nucleotide sequences, ten or more nucleotide sequences, eleven or more nucleotide sequences, or twelve or more nucleotide sequences) that are each at least about 70% identical (e.g, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to any one of two or more native sequences (e.g., native sequences from the human genome). In some embodiments, the engineered 3' UTR comprises two nucleotide sequences that are each at least about 70% identical to any one of two native sequences. In some embodiments, the engineered 3' UTR comprises three nucleotide sequences that are each at least about 70% identical to any one of three native sequences. In some embodiments, the engineered 3' UTR comprises four nucleotide sequences that are each at least about 70% identical to any one of four native sequences. In some embodiments, the engineered 3' UTR comprises five nucleotide sequences that are each at least about 70% identical to any one of five native sequences. In some embodiments, the engineered 3' UTR comprises six nucleotide sequences that are each at least about 70% identical to any one of six native sequences. In some embodiments, the engineered 3' UTR comprises seven nucleotide sequences that are each at least about 70% identical to any one of seven native sequences. In some embodiments, the engineered 3' UTR comprises eight nucleotide sequences that are each at least about 70% identical to any one ofeight native sequences. In some embodiments, the engineered 3' UTR comprises nine nucleotide sequences that are each at least about 70% identical to any one of nine native sequences. In some embodiments, the engineered 3' UTR comprises ten nucleotide sequences that are each at least about 70% identical to any one of ten native sequences. In some embodiments, the engineered 3' UTR comprises eleven nucleotide sequences that are each at least about 70% identical to any one of eleven native sequences. In some embodiments, the engineered 3' UTR comprises twelve nucleotide sequences that are each at least about 70% identical to any one of twelve native sequences. In some embodiments, the engineered 3' UTR comprises more than twelve nucleotide sequences that are each at least about 70% identical to any one of more than twelve native sequences.

[0071] In some embodiments, the two or more engineered 3' UTR nucleotide sequences are non-identical. In some embodiments, each of the two or more engineered 3' UTR nucleotide sequences corresponds to (i.e. , is at least about 70% identical to) a different native sequence of the two or more native sequences. In some embodiments, the two or more engineered 3' UTR nucleotide sequences are arranged in the same order relative to the two or more corresponding native sequences. In some embodiments, the two or more engineered 3' UTR nucleotide sequences are arranged in a different order relative to the two or more corresponding native sequences. In some embodiments, the two or more engineered 3' UTR nucleotide sequences are separated by a different intervening nucleotide sequence relative to the intervening nucleotide sequence separating the two or more corresponding native sequences. In some embodiments, the two or more engineered 3' UTR nucleotide sequences are directly linked.

[0072] In some embodiments, the native sequence or sequences are each from a gene (e.g. , from a human gene). In some embodiments, the two or more native sequences are each from the same gene (e.g , the same human gene). In some embodiments, the native sequence or sequences are each from an untranslated region (UTR) of a gene. In some embodiments, the two or more native sequences are each from a UTR of the same gene. In some embodiments, the native sequence or sequences are each from the 3' UTR of a gene. In some embodiments, the two or more native sequences are each from the 3' UTR of the same gene. In some embodiments, the native sequence or sequences are each from a gene that exhibits tissue- and / or cell type-specific expression (e.g., SLC26A4). In some embodiments, the two or more native sequences are each from the same gene that exhibits tissue- and / or cell type-specific expression (e.g., SLC26A4).

[0073] In some embodiments, the native sequence or sequences each exhibit high sequence conservation across mammalian genomes. In some embodiments, the native sequence orsequences each exhibit high sequence conservation across human and non-human primate species genomes. In some embodiments, the native sequence or sequences are each from a region of a genome (e.g, a human genome) that shares high sequence homology with other genomes of interest (e.g., mammalian genomes, human and non-human primate genomes). In some embodiments, the native sequence or sequences are selected from SEQ ID NOs: 3-11.

[0074] In some embodiments, the engineered 3' UTR comprises two or more nucleotide sequences (e.g, three or more nucleotide sequences, four or more nucleotide sequences, five or more nucleotide sequences, six or more nucleotide sequences, seven or more nucleotide sequences, eight or more nucleotide sequences, nine or more nucleotide sequences, ten or more nucleotide sequences, eleven or more nucleotide sequences, or twelve or more nucleotide sequences) that are each at least about 70% identical (e.g. at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to any one of SEQ ID NOs: 3-11.

[0075] In some embodiments, the engineered 3' UTR comprises two or more non-identical nucleotide sequences (e.g.. three or more non-identical nucleotide sequences, four or more nonidentical nucleotide sequences, five or more non-identical nucleotide sequences, six or more non-identical nucleotide sequences, seven or more non-identical nucleotide sequences, eight or more non-identical nucleotide sequences, nine or more non-identical nucleotide sequences, ten or more non-identical nucleotide sequences, eleven or more non-identical nucleotide sequences, or twelve or more non-identical nucleotide sequences) that are each at least about 70% identical (e g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%. at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to any one of SEQ ID NOs: 3-11.

[0076] In some embodiments, the engineered 3' UTR comprises one, two. three, four, five, six, seven, eight, or nine nucleotide sequences (e.g, non-identical nucleotide sequences) selectedfrom: a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 3; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 4; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 5; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 6; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 7 ; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 8; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 9; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 11.

[0077] In some embodiments, the engineered 3' UTR comprises one, two. three, four, five, six, seven, eight, or nine nucleotide sequences (e.g., non-identical nucleotide sequences) selected from: a nucleotide sequence that is at least 70% identical (e g, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 3; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 4; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 5; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 6; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 7; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 8; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 9; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least 70% identical to SEQ ID NO: 11.

[0078] In some embodiments, the engineered 3' UTR comprises, from 5' to 3'. a nucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. at least about 99%. or about 100% identical) to SEQ ID NO: 3; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 4; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 5; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 6; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 7 a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 8; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 9; a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least about 70% identical to SEQ ID NO: 11.

[0079] In some embodiments, the engineered 3' UTR comprises, from 5' to 3', a nucleotide sequence that is at least 70% identical (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:3; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 4; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 5; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 6; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 7; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 8; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 9; a nucleotide sequence that is at least 70% identical to SEQ ID NO: 10; and a nucleotide sequence that is at least 70% identical to SEQ ID NO: 11.

[0080] In some embodiments, the engineered 3' UTR comprises, from 5' to 3', a nucleotide sequence consisting of SEQ ID NO: 3; a nucleotide sequence consisting of SEQ ID NO: 4; a nucleotide sequence consisting of SEQ ID NO: 5; a nucleotide sequence consisting of SEQ ID NO: 6; a nucleotide sequence consisting of SEQ ID NO: 7; a nucleotide sequence consisting of SEQ ID NO: 8; a nucleotide sequence consisting of SEQ ID NO: 9; a nucleotide sequence consisting of SEQ ID NO: 10; and a nucleotide sequence consisting of SEQ ID NO: 11.

[0081] In some embodiments, the engineered 3' UTR comprises a nucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 13. In some embodiments, the engineered 3' UTR comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 13.

[0082] In some embodiments, the engineered 3' UTR comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 14 or 15. In some embodiments, the engineered 3' UTR comprises the nucleotide sequence of SEQ ID NO: 14 or 15. In some embodiments, the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 14 or 15.B. Transgene

[0083] In some embodiments, the polynucleotides provided herein comprise a transgene. In some embodiments, the transgene is positioned 5' of the 3' UTR. In some embodiments, the transgene comprises one or more sequences encoding an RNA molecule. Suitable RNA molecules include, without limitation, microRNAs (miRNAs), small hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), antisense RNAs, antisense oligonucleotides (AON), decoy hairpin RNA oligonucleotide (dONs), guide RNAs (gRNAs), antagomirs, miRNA sponges, RNA aptazymes. RNA aptamers, mRNA, long non-coding RNAs (IncRNAs), ribozymes, and synthetic RNAs known in the art. In some embodiments, the transgene encodes one or more polypeptides, or one or more fragments thereof. Such transgenes can comprise the complete coding sequence of a polypeptide, or only a fragment of a coding sequence of a polypeptide. In some embodiments, the transgene comprises a protein coding sequence (e.g., a pendrin protein coding sequence).

[0084] In some embodiments, the polynucleotides provided herein comprise a protein coding sequence. In some embodiments, the protein coding sequence encodes a wild-tj pe polypeptide (e.g., a wild-type human polypeptide). In some embodiments, the protein coding sequence encodes a polypeptide with one or more alterations relative to a wild-type version of saidpolypeptide. In some embodiments, the protein coding sequence is wild-tj pe (e g., a wild-type human protein coding sequence). In some embodiments, the protein coding sequence comprises one or more alterations relative to a wild-type version of said protein coding sequence. In some embodiments, the protein coding sequence is silently altered relative to a wild-type version of said protein coding sequence. In some embodiments, the protein coding sequence is codon-optimized for expression in a host (e.g, in a human host).

[0085] In some embodiments, the polynucleotides provided herein comprise a pendrin protein coding sequence. In some embodiments, the pendrin protein is a human pendrin protein. In some embodiments, the pendrin protein comprises an amino acid sequence at least about 80% identical (e.g, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 1. In some embodiments, the pendrin protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the pendrin protein consists of the amino acid sequence of SEQ ID NO: 1.

[0086] In some embodiments, the pendrin protein coding sequence comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence at least about 80% identical (e.g, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 1. In some embodiments, the pendrin protein coding sequence comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the pendrin protein coding sequence consists of a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0087] In some embodiments, the pendrin protein coding sequence comprises a nucleotide sequence at least about 70% identical (e.g. , at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 2. In some embodiments, the pendrin protein coding sequence is silently altered with respect to SEQ ID NO: 2. In some embodiments, the pendrin protein coding sequence comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the pendrin protein coding sequence consists of the nucleotide sequence of SEQ ID NO: 2.C. Additional genetic elements

[0088] In some embodiments, the polynucleotides provided herein comprise one or more additional genetic elements. In some embodiments, the one or more additional genetic elements regulate expression of a transgene or protein coding sequence. The additional genetic elements may include, without limitation, an untranslated region (UTR), a promoter, an intron element, an enhancer, a polyadenylation sequence, and / or a posttranscriptional response element.

[0089] In some embodiments, the polynucleotides provided herein compnse a 5' untranslated region (5' UTR). In some embodiments, the 5' UTR is positioned 5' of the engineered 3' UTR. In some embodiments, the 5' UTR is positioned 5' of a transgene. In some embodiments, the 5' UTR is positioned 5' of a protein coding sequence (e.g.. a pendrin protein coding sequence).

[0090] In some embodiments, the 5' UTR has a length of about 5 to about 500 nucleotides (e.g. , about 5 to about 450, about 5 to about 400, about 5 to about 350, about 5 to about 300, about 5 to about 250, about 5 to about 200, about 5 to about 150, about 10 to about 500, about 10 to about 450, about 10 to about 400, about 10 to about 350, about 10 to about 300, about 10 to about 250, about 10 to about 200. about 10 to about 150, about 15 to about 500, about 15 to about 450, about 15 to about 400, about 15 to about 350, about 15 to about 300, about 15 to about 250, about 15 to about 200, or about 15 to about 150 nucleotides). In some embodiments, the 5' UTR is about 15 to about 200 nucleotides in length.

[0091] In some embodiments, the 5' UTR has a length of less than about 500 nucleotides (e.g. , less than about 475, less than about 450, less than about 425, less than about 400, less than about 375, less than about 350, less than about 325, less than about 300, less than about 275, less than about 250, less than about 225, less than about 200, less than about 175, or less than about 150 nucleotides). In some embodiments, the 5' UTR is less than about 200 nucleotides in length.

[0092] In some embodiments, the 5' UTR has a length of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150. about 160, about 170, about 180, about 190, about 200, about220, about 240, about 260, about 280, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 nucleotides. In some embodiments, the 5' UTR is about 30 nucleotides in length. In some embodiments, the 5' UTR is about 35 nucleotides in length. In some embodiments, the 5' UTR is about 40 nucleotides in length.

[0093] In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%. at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a native sequence (e.g, a native sequence from the human genome). In some embodiments, the native sequence is from a gene (e.g. , from a human gene). In some embodiments, the native sequence is from a 5' UTR region of a gene.

[0094] In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%. at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. at least about 99%, or about 100% identical) to a nucleotide sequence set forth in SEQ ID NO: 16.

[0095] In some embodiments, the 5' UTR comprises a nucleotide sequence that is at least about 70% identical (e g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%. at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 17. In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the 5' UTR consists of the nucleotide sequence of SEQ ID NO: 17.

[0096] In some embodiments, the polynucleotides provided herein comprise a promoter. In some embodiments, the promoter is operably linked to a transgene. In some embodiments, the promoter is operably linked to a protein coding sequence (e.g., a pendrin protein coding sequence). In some embodiments, the promoter is positioned 5' of a transgene. In some embodiments, the promoter is positioned 5' of a protein coding sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue- and / or cell type-specific promoter.

[0097] In some embodiments, the promoter comprises a nucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%. at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%. at least about 99%, or about 100% identical) to a native sequence (e.g, a native sequence from the human genome). In some embodiments, the native sequence is from a promoter (e.g. , from a human promoter). In some embodiments, the promoter is a SLC26A4 promoter. In some embodiments, the promoter is a human SLC26A4 promoter.

[0098] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV), long terminal repeat (LTR) promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (See, e.g., Boshart etal., Cell, 41 :521-530 (1985)) the simian vacuolating virus 40 (SV40) promoter, the dihydrofolate reductase promoter, the CAG promoter (See. e g., Miyazaki et al., 1989, Gene 79(2):269-277), the [3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the elongation factor 1 -alpha 1 (EFla) promoter. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter. In some embodiments, the promoter is an enhanced chicken beta-actin promoter. In some embodiments, the CBA promoter comprises a nucleotide sequence that is at least about 70% identical (e.g , at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. or about 100% identical) to SEQ ID NO: 18 or 19. In some embodiments, the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 18 or 19. In some embodiments, the CBA promoter consists of the nucleotide sequence of SEQ ID NO: 18 or 19.

[0099] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other promoters have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)). the tetracycline-inducible system (Gossen et al., Science. 268: 1766- 1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486- inducible system (Wang et al., Nat. Biotech. , 15:239-243 (1997) and Wang et al., Gene Ther. , 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest. , 100:2865-2872 (1997)).

[0100] In some embodiments, the promoter comprises a nucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to any one of SEQ ID NOs: 20- 26. In some embodiments, the CBA promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 20-26. In some embodiments, the CBA promoter consists of the nucleotide sequence of any one of SEQ ID NOs: 20-26.

[0101] In some embodiments, the polynucleotides provided herein comprise an intron element. In some embodiments, the intron element is positioned between a promoter and a transgene. In some embodiments, the intron element is positioned between a promoter and aprotein coding sequence. Such intron elements can increase transgene expression, for example, by reducing transcriptional silencing and enhancing mRNA export from the nucleus to the cytoplasm.

[0102] In some embodiments, the intron element comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a native sequence (e.g, a native sequence from the human genome). In some embodiments, the native sequence is from an intron (<?.g, from a human intron). In some embodiments, the intron element is a SLC26A4 intron element. In some embodiments, the intron element is a human SLC26A4 intron element.

[0103] In some embodiments, the intron element is an exogenous intron element (e.g., comprising at least an intron sequence from a different species or a different gene from the same species, and / or a synthetic intron sequence). In some embodiments, the intron element is an exogenous intron element comprising at least a portion of an intron sequence from a different species. In some embodiments, the intron element is an exogenous intron element comprising at least a portion of an intron sequence from a different gene from the same species. In some embodiments, the intron element is an exogenous intron element comprising a synthetic intron sequence. In some embodiments, the intron element is an exogenous intron element comprising a combination of at least an intron sequence from a different species or a different gene from the same species, and / or a synthetic intron sequence.

[0104] A skilled worker will appreciate that intron elements can be designed to mediate RNA splicing by introducing any consensus splicing motifs known in the art (e.g, in Sibley et al., 2016, Nature Reviews Genetics, 17:407-21, which is incorporated by reference herein in its entirety). Exemplary intron sequences are provided in Lu et al., 2013, Molecular Therapy 21(5):954-63, and Lu et al., 2017 Hum. Gene Ther. 28(1): 125-34, which are incorporated by reference herein in their entirety.

[0105] In some embodiments, the polynucleotides provided herein comprise an exogenous intron element. In some embodiments, the intron element comprises an SV40 intron element. In some embodiments, the intron element comprises a CBA intron element.

[0106] In some embodiments, the intron element comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 27. In some embodiments, the intron element comprises the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the intron element consists of the nucleotide sequence of SEQ ID NO: 27.

[0107] In some embodiments, the polynucleotides provided herein comprise an enhancer. In some embodiments, the enhancer is positioned 5' of a promoter. In some embodiments, the enhancer comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a native sequence (e.g.. a native sequence from the human genome). In some embodiments, the native sequence is from an enhancer (e.g, from a human enhancer). In some embodiments, the enhancer is a SLC26A4 enhancer. In some embodiments, the enhancer is a human SLC26A4 enhancer.

[0108] In some embodiments, the enhancer is an exogenous enhancer (e.g, comprising at least an enhancer sequence from a different species or a different gene from the same species, and / or a synthetic enhancer sequence). In some embodiments, the enhancer is an exogenous enhancer comprising at least a portion of an enhancer sequence from a different species. In some embodiments, the enhancer is an exogenous enhancer comprising at least a portion of an enhancer sequence from a different gene from the same species. In some embodiments, the enhancer is an exogenous enhancer comprising a synthetic enhancer sequence. In some embodiments, the enhancer is an exogenous enhancer comprising a combination of at least an enhancer sequence from a different species or a different gene from the same species, and / or a synthetic enhancer sequence.

[0109] In some embodiments, the polynucleotides provided herein comprise an exogenous enhancer. In some embodiments, the enhancer comprises a CMV enhancer. In some embodiments, the enhancer comprises an RSV enhancer.

[0110] In some embodiments, the enhancer comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%. at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%. at least about 99%, or about 100% identical) to SEQ ID NO: 28. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the enhancer consists of the nucleotide sequence of SEQ ID NO: 28.

[0111] In some embodiments, the polynucleotides provided herein comprise one or more conventional expression control elements which are operably linked with a transgene or protein coding sequence in a manner that permits transcription, translation, and / or expression of the transgene or protein coding sequence in a cell comprising the polynucleotide (e.g, in a cell transfected with a vector provided herein). Expression control elements include, without limitation, appropriate transcription initiation signals; termination signals; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g. Kozak consensus sequence); and sequences that enhance protein stability .

[0112] In some embodiments, the polynucleotides provided herein comprise a polyadenylation sequence. In some embodiments, the polyadenylation sequence is positioned 3' of the engineered 3' UTR. In some embodiments, the polyadenylation sequence comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to a native sequence (e.g. , a native sequence from the human genome). In some embodiments, the native sequence is a poly adenylation sequence (e.g, a human polyadenylation sequence).In some embodiments, the poly adenylation sequence is an SLC26A4 polyadenylation sequence. In some embodiments, the polyadenylation sequence is a human SLC26A4 polyadenylation sequence.

[0113] In some embodiments, the poly adenylation sequence is an exogenous polyadenylation sequence (e.g., comprising at least a polyadenylation sequence from a different species or a different gene from the same species, and / or a synthetic polyadenylation sequence). In some embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence comprising at least a portion of a polyadenylation sequence from a different species. In some embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence comprising at least a portion of a polyadenylation sequence from a different gene from the same species. In some embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence comprising a synthetic polyadenylation sequence. In some embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence comprising a combination of at least a polyadenylation sequence from a different species or a different gene from the same species, and / or a synthetic polyadenylation sequence.

[0114] In some embodiments, the poly adenylation sequence comprises a bovine growth hormone polyadenylation sequence. In some embodiments, the polyadenylation sequence comprises an SV40 polyadenylation sequence.

[0115] In some embodiments, the polyadenylation sequence comprises a nucleotide sequence that is at least about 70% identical (e.g.. at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 29. In some embodiments, the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the polyadenylation sequence consists of the nucleotide sequence of SEQ ID NO: 29.

[0116] In some embodiments, the polynucleotides provided herein comprise a posttranscriptional response element. In some embodiments, the posttranscriptional response element is a woodchuck hepatitis virus posttranscriptional regulatory' element (WPRE), a mouse RNA transport element (RTE), a constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MPMV), or the 5'untranslated region of the human heat shock protein 70 (Hsp70 5' UTR). In some embodiments, the posttranscriptional response element comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 30. In some embodiments, the posttranscriptional response element comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the posttranscriptional response element consists of the nucleotide sequence of SEQ ID NO: 30.

[0117] In some embodiments, the polynucleotides provided herein comprise a sequence encoding a polypeptide tag that is useful for, e.g. , purification of a linked polypeptide. In some embodiments, the sequence encoding a polypeptide tag is positioned such that the tag is linked to a polypeptide (e.g, a pendrin protein), e.g.. at a 3' or 5' terminal end of the polypeptide. In some embodiments, the sequence encoding a polypeptide tag is positioned 3' of a transgene or coding sequence. In some embodiments, the polypeptide tag is a human influenza hemagglutinin (HA) tag. In some embodiments, a sequence encoding an HA tag comprises a nucleotide sequence that is at least about 70% identical (e.g, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 31. In some embodiments, the sequence encoding an HA tag comprises the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the sequence encoding an HA tag consists of the nucleotide sequence of SEQ ID NO: 31.D. Vectors

[0118] In one aspect, the present disclosure provides a vector comprising a polynucleotide as described herein. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector is a gene therapy vector. A gene therapy vector may be a viral vector(e.g, a lentiviral vector, an adeno-associated virus vector, an adenoviral (Ad) vector, etc.), a plasmid, a closed-ended DNA (e.g., ceDNA), a lipid / DNA nanoparticle, etc. In some embodiments, a gene therapy vector is a viral vector. In some embodiments, a polynucleotide provided herein comprises one or more viral replication sequences, e.g. , lentiviral long terminal repeats (LTRs), adeno-associated vims (AAV) inverted terminal repeats (ITRs), etc.

[0119] In some embodiments, the polynucleotides provided herein comprise an inverted terminal repeat (ITR) positioned at the 5' end (5' ITR). In some embodiments, the polynucleotides provided herein comprise an inverted terminal repeat (ITR) positioned at the 3' end (3' ITR). In some embodiments, the polynucleotides provided herein comprise a 5' ITR and a 3' ITR. Generally, ITR sequences are about 145 bp in length. In some embodiments, the polynucleotides provided herein comprise substantially an entire ITR sequence. In some embodiments, some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of one in the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989): and K. Fisher et al., J Virol., 70:520 532 (1996)).

[0120] In some embodiments, the 5' ITR and / or the 3' ITR is an AAV ITR or a variant thereof. The AAV ITR may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, a polynucleotide provided herein comprises a 5' ITR and / or a 3' ITR derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4. AAV5, AAV6, AAV6.2, AAV7, AAV8. AAV9, AAV 10, AAV 11, and variants thereof. In some embodiments, a polynucleotide provided herein comprises a 5' ITR and / or a 3' ITR derived from AAV2.

[0121] In some embodiments, the 5' ITR comprises anucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 32. In some embodiments, the 5' ITR comprises the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the 5' ITR consists of the nucleotide sequence of SEQ ID NO: 32.

[0122] In some embodiments, the 3' ITR comprises anucleotide sequence that is at least about 70% identical (e.g., at least about 71%, at least about 72%, at least about 73%, at least about74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%. at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to SEQ ID NO: 33. In some embodiments, the 3' ITR comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the 3' ITR consists of the nucleotide sequence of SEQ ID NO: 33.

[0123] In some embodiments, the 5' ITR nucleotide sequence and the 3' ITR nucleotide sequence are substantially complementary to each other (e.g., are complementary to each other except for mismatch at 1, 2, 3, 4, or 5 nucleotide positions in the 5' or 3' ITR).

[0124] In some embodiments, the 5' ITR and / or the 3' ITR is modified to reduce or abolish resolution by Rep protein (“non-resolvable ITR”). In some embodiments, the non-resolvable ITR comprises an insertion, deletion, or substitution in the nucleotide sequence of the terminal resolution site. Such modification allows formation of a self-complementary, double-stranded DNA genome of the AAV after the rAAV genome is replicated in an infected cell. Exemplary non-resolvable ITR sequences are known in the art (see e.g., those provided in U.S. Patent Nos. 7,790,154 and 9,783,824, which are incorporated by reference herein in their entirety).E. Cell-type specific expression

[0125] In some embodiments, a polynucleotide provided herein is capable of expressing a transgene (e.g. , a pen drin protein coding sequence) in a tissue- and / or cell type-specific manner. In some embodiments, the polynucleotide is capable of expressing a transgene (e.g., a pendrin protein coding sequence) in cells that normally express the SLC26A4 gene. In some embodiments, the polynucleotide is capable of expressing a transgene (e.g., a pendrin protein coding sequence) in inner ear cells. In some embodiments, the polynucleotide is capable of expressing a transgene (e.g., a pendrin protein coding sequence) in cochlear cells. In some embodiments, the polynucleotide is capable of expressing a transgene (e.g., a pendrin protein coding sequence) in root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.F. Exemplary polynucleotides

[0126] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': a pendrin protein coding sequence, and an engineered 3' UTR. In some embodiments, the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 and / or the engineered 3' UTR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-15. In some embodiments, the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2 and / or the engineered 3' UTR comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 13-15. In some embodiments, a polynucleotide provided herein comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 34-36. In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence any one of SEQ ID NOs: 34-36.

[0127] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': a 5' UTR. a pendrin protein coding sequence, and an engineered 3' UTR. In some embodiments, the 5' UTR is at least 80%, 81%, 82%, 83%. 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; and / or the engineered 3' UTR is at least 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-15. In some embodiments, the 5' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 17; the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2; and / or the engineered 3' UTR comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 13-15. In some embodiments, a polynucleotide provided herein comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 37-39. In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence any one of SEQ ID NOs: 37-39.

[0128] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': a promoter, a 5' UTR, a pendrin protein coding sequence, an engineered 3' UTR, and a polyadenylation sequence. In some embodiments, the promoter is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 18-26; the 5' UTR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; the engineered 3' UTR is at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-15; and / or the polyadenylation sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29. In some embodiments, the promoter comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 18-26; the 5' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 17; the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2; the engineered 3' UTR comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 13-15; and / or the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the polynucleotide further comprises one or more of: an enhancer positioned 5' of the promoter: an intron element positioned between the promoter and the 5' UTR; and / or a posttranscriptional response element positioned between the engineered 3' UTR and the polyadenylation sequence. In some embodiments, the enhancer is at least 80%, 81%. 82%. 83%. 84%. 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; the intron element is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; and / or the posttranscriptional response element is at least 80%, 81%, 82%. 83%. 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30. In some embodiments, the enhancer comprises or consists of the nucleotide sequence of SEQ ID NO: 28; the intron element comprises or consists of the nucleotide sequence of SEQ ID NO: 27; and / or the posttranscriptional response comprises or consists of the nucleotide sequence of SEQ ID NO: 30.

[0129] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': a 5' ITR; a promoter, a 5' UTR, a pendrin protein coding sequence, an engineered 3' UTR, a polyadenylation sequence and a 3' ITR. In some embodiments, the 5' ITR is at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32; the promoter is at least 80%, 81%, 82%, 83%, 84%,85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 18-26; the 5' UTR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; the engineered 3' UTR is at least 80%, 81%, 82%, 83%, 84%. 85%. 86%. 87%. 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-15; the polyadenylation sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and / or the 3' ITR is at least 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the 5' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 32; the promoter comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 18-26; the 5' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 17; the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2; the engineered 3' UTR comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 13-15; the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29; and / or the 3' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide further comprises one or more of: an enhancer positioned between the 5' ITR and the promoter; an intron element positioned between the promoter and the 5' UTR; and / or a posttranscriptional response element positioned between the engineered 3' UTR and the polyadenylation sequence. In some embodiments, the enhancer is at least 80%, 81%, 82%, 83%, 84%, 85%. 86%. 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; the intron element is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; and / or the posttranscriptional response element is at least 80%, 81%, 82%, 83%, 84%. 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% identical to SEQ ID NO: 30. In some embodiments, the enhancer comprises or consists of the nucleotide sequence of SEQ ID NO: 28; the intron element comprises or consists of the nucleotide sequence of SEQ ID NO: 27; and / or the posttranscriptional response comprises or consists of the nucleotide sequence of SEQ ID NO: 30.

[0130] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': an enhancer, a promoter, an intron element, a 5' UTR, a pendrin protein coding sequence, anengineered 3' UTR, and a polyadenylation sequence. In some embodiments, the enhancer is at least 80%, 81%, 82%, 83%, 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; the promoter is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; the intron element is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; the 5' UTR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; the engineered 3' UTR is at least 80%. 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and / or the polyadenylation sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29. In some embodiments, the enhancer comprises or consists of the nucleotide sequence of SEQ ID NO: 28; the promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 19; the intron element comprises or consists of the nucleotide sequence of SEQ ID NO: 27; the 5' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 17; the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2; the engineered 3' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 13; and / or the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the polynucleotide comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO: 41.

[0131] In some embodiments, a polynucleotide provided herein comprises from 5' to 3': a 5' ITR, an enhancer, a promoter, an intron element, a 5' UTR, a pendrin protein coding sequence, an engineered 3' UTR, a polyadenylation sequence, and a 3' ITR. In some embodiments, the 5' ITR is at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32; the enhancer is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; the promoter is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; the intron element is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27: the 5' UTR is at least 80%, 81%. 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; the pendrin protein coding sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; the engineered 3' UTR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%, 96%. 97%, 98%, or 99% identical to SEQ ID NO: 13; the polyadenylation sequence is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and / or the 3' ITR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the 5' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 32; the enhancer comprises or consists of the nucleotide sequence of SEQ ID NO: 28; the promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 19; the intron element comprises or consists of the nucleotide sequence of SEQ ID NO: 27; the 5' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 17; the pendrin protein coding sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2; the engineered 3' UTR comprises or consists of the nucleotide sequence of SEQ ID NO: 13; the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29; and / or the 3' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO: 42.III. Recombinant Adeno-Associated Viruses (rAAVs)

[0132] In one aspect, the present disclosure provides recombinant AAVs (rAAVs) comprising a capsid protein and a polynucleotide (e.g, an AAV vector) described herein.

[0133] Methods for obtaining recombinant AAVs having a desired capsid protein are known in the art. (See. for example, US 2003 / 0138772, which is incorporated herein by reference). In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa. about 72 kDa, andabout 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome, and / or interact with the host. In some embodiments, capsid proteins increase delivery of the viral genome to a host in a tissue- or cell-ty pe specific manner. In some embodiments, the presently disclosed rAAVs comprise a capsid protein that increases delivery of the viral genome to cells in the inner ear.

[0134] In some embodiments, the rAAVs provided herein comprise an AAV capsid protein of an AAV serotype selected from AAV9.PHP.B, AAV9.PHP.eB, exoAAV, Anc80, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, and AAV-S. AAV2.7m8 is capable of delivering a transgene targeting cochlear hair cells and supporting cells and the retina. AAV2.7m8 shows good transduction to the inner ear (Isgrig et al., ”AAV2.7m8 is apowerful viral vector for inner ear gene therapy, ” Nature Communications volume 10, Article number: 427 (2019)). In some embodiments, the capsid protein is of AAV seroty pe 9 (AAV9). In some embodiments, an AAV capsid protein is of a seroty pe derived from AAV9 (e.g., an AAV9 capsid variant), for example, AAV9.PHP.B. In some embodiments, the AAV9 capsid variant is AAV9.PHP.B. In some embodiments, the AAV9 capsid variant is AAV-S. AAV-S is an AAV9 capsid protein variant originally developed for targeting central nervous system (CNS) (Hanlon et al., Selection of an Efficient AAV Vector for Robust CNS Transgene Expression, Molecular Therapy Method & Clinical Development, vol. 15, pp. 320-332, December 13, 2019, and PCT / US2020 / 025720, which are incorporated herein by reference). Surprisingly, AAV-S showed good transducing efficiency for inner ear cells, (see., e.g., Hanlon et al., AAV-S: A novel AAV vector selected in brain transduces the inner ear with high efficiency, Molecular Therapy Vol 18 No 4S1, April 28, 2020, Abstract 151, which is incorporated herein by reference), including, but not limited to: outer hair cells (OHCs), inner hair cells (IHCs), supporting cells (e.g., border cell, inner phalangeal cell, inner pillar cell, outer pillar cell, Deiters’ cell, Hensen’s, or Claudius’ cell), spiral ganglion neuron, spiral limbus cells (e.g. , glial cell or interdental cell), outer sulcus cells, lateral wall, stria vascularis (e.g., basal cell and intermediate cell), inner sulcus, spiral ligament (e.g., fibrocytes), or cells of the vestibular system. In some embodiments, the AAV capsid is AAV-S. An exemplary amino acid sequence for AAV-S is set forth in SEQ ID NO: 43. In some embodiments, the AAV capsid is an exoAAV. An exoAAV refers to an exosome-associated AAV. An exoAAV capsid protein may be selected from the group consisting of AAV 1 , AAV2, AAV3. AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, and AAV.PHP.B. In some examples, the exoAAV is exoAAVl or exoAAV9.

[0135] The skilled artisan will also realize that conservative amino acid substitutions may be made to provide functionally equivalent variants or homologs of any of the proteins or polypeptides described herein (e.g., a pendrin protein, a capsid protein). As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g.. Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds.. Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T: (f) Q, N; and (g) E, D.

[0136] In some embodiments, an rAAV provided herein is a single stranded AAV (ssAAV). An ssAAV, as used herein, refers to an rAAV with the transgene sequence and its complementary sequence on separate strands and packaged in separate viral capsids. In some embodiments, the rAAV is a self-complementary AAV (scAAV). An scAAV, as used herein, refers to an rAAV with both the transgene sequence and its complementary sequence present on the single strand of an AAV genome. The coding region of an scAAV was designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription.

[0137] In some embodiments, an rAAV provided herein is capable of delivering a transgene (e.g., a pendrin protein coding sequence as described herein) to a mammal. In some embodiments, the mammal is a human or a non-human mammal, such as a mouse, a rat, or a non-human primate (e.g., cynomolgus monkey), a cat, a dog, a pig, a horse, a donkey, a camel, a sheep, or a goat. In some embodiments, the mammal is a human.

[0138] In some embodiments, an rAAV provided herein is capable of delivering a transgene (e.g., a pendrin protein coding sequence as described herein) to the ear. In some embodiments, the rAAV is capable of delivering a transgene to cells in the inner ear (e.g., cochlea, saccule, utricle, and semicircular canals). Non-limiting examples of the inner ear target cells are outer hair cells (OHC), inner hair cells (IHC), spiral ganglion neurons, cells of stria vascularis, cells of inner sulcus, cells of spiral ligament, cells of vestibular system, organ of Corti supportingcells (e.g., epithelial cells of the inner and outer sulcus, and interdental cells), interdental cells in the spiral limbus, root cells within the spiral ligament, pillar cells. Deiters’ cells, Hensen’s cells, Claudius cells, inner phalangeal cells; and border cells, strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal cells of the stria vascularis, fibrocytes in the spiral ligament, fibrocytes in the spiral limbus, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, and supralimbal dark cells. In some embodiments, the rAAV is capable of delivering a transgene to cells that normally express the SLC26A4 gene. In some embodiments, the rAAV has tropism for a subset of inner ear cells (e.g, inner ear cells that normally express the SLC26A4 gene). In some embodiments, the rAAV has tropism for root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells. In some embodiments, the rAAV has tropism for cells of the cochlea.

[0139] In some embodiments, as demonstrated in the Examples herein, rAAVs provided herein comprising an AAV capsid that increases transduction efficiency for inner ear cells (e.g., AAV-S) and a polynucleotide comprising a pendrin protein coding sequence and an engineered 3' UTR (e.g. as described herein) are able to drive highly specific expression of pendrin in a subset of inner ear cells (e.g, inner ear cells that normally express the SLC26A4 gene). Such rAAVs are particularly advantageous in that they reduce toxicity and / or unwanted side effects associated with promiscuous pendrin expression.

[0140] The rAAVs provided herein may be produced using any suitable method available in the art. For example, the rAAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650, which is incorporated herein by reference). Typically, rAAVs are produced by transfecting a host cell with an rAAV vector (e.g, as described herein) to be packaged into rAAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector comprises the “AAV helper function” sequences (e.g.. rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, both of which are incorporated herein by reference. The accessory function vector comprises nucleotide sequences for non-AAV derived viral and / or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions required for AAVreplication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing. AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

[0141] The components to be cultured in a host cell to package an rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g, rAAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0142] The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate construct (e.g, vector). The selected construct may be delivered by any suitable method, including those described herein and known in the art. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g.. Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are known in the art, and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher etal., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745, each of which is incorporated herein by reference.IV. Cells and compositions

[0143] In another aspect, the present disclosure provides cells comprising a polynucleotide, vector or rAAV described herein. In some embodiments, the cell is a mammalian cell (e.g, a human cell), a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In someembodiments, the cell is or has been transfected with a polynucleotide or vector described herein. In some embodiments, the cell is or has been transduced with an rAAV described herein.

[0144] In another aspect, the present disclosure provides compositions comprising a polynucleotide, vector, rAAV, or cell described herein and a pharmaceutically acceptable excipient. Suitable excipients may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. In some embodiments, the pharmaceutically acceptable excipient is compatible with the mode of administration. Further discussion of selection of pharmaceutically acceptable excipients appropriate to particular indications and / or modes of administration is included herein below; In some embodiments, the pharmaceutically acceptable excipient comprises saline, which may be formulated with a variety of buffering solutions (e.g, phosphate buffered saline). Other exemplary excipients include, without limitation, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.

[0145] In some embodiments, a composition provided herein comprises a buffer agent. A buffer agent is a weak acid or base used to maintain the pH of a solution near a chosen value after the addition of another acid or base. In some embodiments, the buffer agent is capable of maintaining physiological pH despite changes in carbon dioxide concentration (e.g., produced by cellular respiration). Exemplary' buffer agents include, but are not limited to, HEPES (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid) buffer, Dulbecco’s phosphate-buffered saline (DPBS) buffer, or phosphate-buffered saline (PBS) buffer. Such buffers may comprise disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[0146] In some embodiments, a composition provided herein further comprises one or more additional pharmaceutical ingredients, e.g., preservatives or chemical stabilizers. Suitable preservatives include, without limitation, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include, without limitation gelatin and albumin.

[0147] In some embodiments, a composition provided herein further comprises one or more surface-active agents, e.g., a surfactant. Surfactants are compounds that lower the surface tension (or interfacial tension) betw een tw o liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Suitable surfactants include, without limitation, non-ionic agents, such as polyoxyethylenesorbitans (e.g.. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g.,Span™ 20, 40, 60, 80 or 85). In some embodiments, compositions with a surface-active agent comprise from 0.05 to 5% surface-active agent, e.g , from 0.1 to 2.5%.

[0148] In some embodiments, a composition provided herein further comprises one or more other ingredients, e.g., mannitol or other pharmaceutically acceptable vehicles.

[0149] In some embodiments, rAAV compositions provided herein are formulated to reduce aggregation of rAAV particles in the composition, particularly where high rAAV concentrations are present (e.g.. ~1013GC / ml or more). Appropriate methods for reducing aggregation may be used, including, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright el al., Molecular Therapy (2005) 12, 171— 178, the contents of which are incorporated herein by reference.)

[0150] In some embodiments, an rAAV composition provided herein comprises an rAAV described herein alone or in combination with one or more other viruses (e.g.. a second rAAV comprising one or more different transgenes). In some embodiments, the rAAV composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each comprising one or more different transgenes.V. Methods of use

[0151] In one aspect, the present disclosure provides methods for specifically expressing pendrin in cells that normally express the SLC26A4 gene in a subject. In another aspect, the present disclosure provides methods for treating hearing loss (e.g. , hearing loss associated with an enlarged vestibular aqueduct, genetic hearing loss, etc.) in a subject in need thereof. In some embodiments, the hearing loss is associated with a mutation in the SLC26A4 gene. In another aspect, the present disclosure provides methods for treating a pendrin-associated disease (e.g., Pendred syndrome or autosomal recessive nonsyndromic deafness 4) in a subject in need thereof. The methods provided herein comprise administering to the subject an effective amount of a polynucleotide described herein or of a vector, rAAV, cell, or composition comprising a polynucleotide described herein. Various embodiments of the provided methods will be detailed below.

[0152] In some embodiments of the provided methods, the subject is a mammal. In some examples, the subject is a human. In other embodiments, the subject is a non-human mammal, such as a mouse, rat, cow, goat, pig, camel, or non-human primate (e.g., cynomolgus monkey). The subject may be at any stage of development and of any gender.

[0153] In some embodiments, the subject has or is suspected to have hearing loss (e.g. , genetic hearing loss). In some embodiments, the hearing loss is associated with a mutation in theSLC26A4 gene. In some embodiments, the mutation in the SLC26A4 gene is a point mutation, a missense mutation, a nonsense mutation, a deletion, an insertion, or a combination thereof. A mutation, as used herein, refers to a substitution of a residue within a sequence, e.g. , a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. The SLC26A4 mutation in a subject (e.g., a subject having or suspected of having genetic hearing loss associated with a deletion or mutation of the SLC26A4 gene) may be identified from a sample obtained from the subject (e.g. , a DNA sample, RNA sample, blood sample, or other biological sample) by any method known in the art. For example, in some embodiments, a nucleic acid (e.g, DNA, RNA, or a combination thereof) is extracted from a biological sample obtained from a subject and nucleic acid sequencing is performed in order to identify a mutation in the SLC26A4 gene. In some embodiments, a mutation in the SLC26A4 gene is detected indirectly, e.g., by quantifying pendrin protein expression (e.g, by Western blot) or function (e.g. , by analyzing structure, function, etc.), or by direct sequencing of the DNA and comparison of the sequence obtained to a control DNA sequence (e.g., a wild-type SLC26A4 DNA sequence). In some embodiments, the mutation in the SLC26A4 gene comprises a mutation of the leucine at residue 236 (e.g. , substitution of the leucine with proline, z.e. , L236P).

[0154] In some embodiments, the subject has or is suspected to have an enlarged vestibular aqueduct (EVA). In some embodiments, the subject is or has been diagnosed with EVA. In some embodiments, the subject has or is suspected to have Pendred syndrome (PDS). In some embodiments, the subject is or has been diagnosed with PDS. In some embodiments, the subject has or is suspected to have autosomal recessive nonsyndromic deafness 4 (DFNB4). In some embodiments, the subject is or has been diagnosed with DFNB4.

[0155] In some embodiments, an effective amount of a polynucleotide described herein (or of a vector, rAAV, cell, or composition comprising a polynucleotide described herein) is an amount sufficient to transfect (or infect in the context of rAAV mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, the target tissue is cochlear. In some embodiments, the target cells are cells that normally express the SLC26A4 gene. In some embodiments, the target cells comprise root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells. In some embodiments, an effective amount of a polynucleotide is an amount sufficient to have a therapeutic benefit in a subject, e.g., to increase or supplement the expression of a gene or protein of interest (e.g., pendrin protein), to improve in the subject one or more symptoms of a disease (e.g., a symptomor sign of PDS or DFNB4), etc. The effective amount will depend on a variety of factors, such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subjects and tissue as described elsewhere in the disclosure. In some embodiments, an effective amount of an rAAV may be an amount sufficient to produce a stable somatic transgenic animal model. In some embodiments, multiple doses of an rAAV are administered.

[0156] The dose of rAAV required to achieve a particular therapeutic effect, e.g, the units of dose in viral genome copies per kilogram of body weight (GC / kg or VG / kg), will vary based on several factors including, but not limited to: the route of rAAV administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or rAAV product. One of skill in the art can readily determine an rAAV dose range to treat a patient having a particular disease or disorder (e.g., a pendrin-associated disease) based on the aforementioned factors, as well as other factors.

[0157] An effective amount of an rAAV described herein may depend on the rAAV used. The invention is based in part on the recognition that an rAAV comprising an AAV capsid that increases transduction efficiency for inner ear cells (e.g., AAV-S) and a polynucleotide comprising a pendrin protein coding sequence and an engineered 3' UTR (e.g., as described herein) mediate highly efficient transduction of cochlear tissue and result in highly tissue- and cell type-specific transgene expression.

[0158] An effective amount of an rAAV described herein may also depend on the mode of administration. For example, targeting cochlear tissue by injection through the round window membrane of the inner ear may require different (e.g., higher or lower) doses, in some cases, than targeting cochlear tissue by another method (e.g, systemic administration, topical administration).

[0159] In some embodiments, a polynucleotide described herein, or a vector, rAAV, cell, or composition comprising a polynucleotide described herein, (e.g., an rAAV encoding pendrin) is administered to a subject (e.g., a subject with pendrin-associated diseases) in combination with other known treatment methods for hearing loss and / or pendrin-associated diseases.

[0160] The methods provided herein comprise administering to a subject an effective amount of a composition described herein (or of a polynucleotide, vector, rAAV, or cell described herein). Administration may be via any suitable route. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery’ to the selected organ (e.g, the ear) or tissue, intravenous, intramuscular, subcutaneous, intradermal.intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

[0161] In some embodiments, the administration is via injection. In some embodiments, the injection is through the round window membrane of the cochlea, into the scala media of the cochlea, into the scala vestibuli of the cochlea, into a semicircular canal of the inner ear, or into the saccule or the utricle of the inner ear. In some embodiments, the injection is injection through the round window membrane of the inner ear. In some embodiments, the injection is posterior semicircular canal injection. In some embodiments, the administration is via topical administration (<?.g., topical administration to an ear).

[0162] A polynucleotide described herein (or vector. rAAV, cell, or composition comprising a polynucleotide described herein) may be delivered to a subject in compositions according to any appropriate method known in the art. In some embodiments, an rAAV described herein is suspended in a pharmaceutically acceptable carrier (z. e.. in a composition) and administered to a subject, e.g., host animal, patient, experimental animal.

[0163] A polynucleotide described herein (or vector. rAAV, cell, or composition comprising a polynucleotide described herein) can be delivered to any organ or tissue of interest in a subj ect. In some embodiments, an rAAV described herein is delivered to the inner ear. Delivery of the rAAV to a mammalian subject may be via, for example, injection to the ear. In some embodiments, the injection is to the ear through the round window membrane of the inner ear, into the scala media of the cochlea, into the scala vestibuli of the cochlea, into a semicircular canal of the inner ear, or into the saccule or the utricle of the inner ear. In some embodiments, the rAAV is delivered to the ear by topical administration (e.g., ear drops). In some embodiments, the injection is not topical administration. Combinations of administration methods (e.g, topical administration and injection through round window membrane of the inner ear) can also be used.

[0164] In some embodiments, rAAVs in suitably formulated compositions (e.g., as described herein) are delivered directly to target tissue, e.g., directly to inner ear tissue. In some embodiments, it is desirable to deliver the rAAV -based therapeutic constructs via another route either separately or in addition, e.g., subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, orally, or intraperitoneally. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver an rAAV described here.

[0165] Compositions suitable for injectable use may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile. It must be stable under the conditions of manufacture and storage and must be preserved to prevent contamination with microorganisms, such as bacteria, fungi, and other viruses. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), 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 dispersion and by the use of surfactants. The prevention of contamination by microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or salts (e.g. , sodium chloride). Prolonged absorption of the injectable composition can be achieved by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0166] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary', and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, and injection through the round window membrane of the inner ear. In this respect, a suitable sterile aqueous medium may be employed. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see for example, Remington ’s Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject / host.

[0167] Sterile injectable solutions are prepared by incorporating an rAAV described herein in the required amount in the appropriate solvent with various of the other ingredients described herein, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-dry ing and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0168] The compositions provided herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts include but are not limited to hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0169] Delivery7vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable cells in a subject. In some embodiments, a polynucleotide provided herein is formulated for delivery encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle, or the like. Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the polynucleotide or rAAV vectors provided herein. Methods for the formation and use of liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516, which is incorporated herein by reference). Further, various methods of liposome and liposome-like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587, each of which is incorporated herein by reference).

[0170] In some embodiments, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, ultrafine particles (sized around 0. 1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0171] In another aspect, the present disclosure provides a polynucleotide, vector, rAAV, cell, or composition described herein for use in a method for specifically expressing pendrin in cells that normally express the SLC26A4 gene in a subject. In another aspect, the present disclosure provides a polynucleotide, vector. rAAV. cell, or composition described herein for use in a method for treating genetic hearing loss in a subject in need thereof. In some embodiments, thegenetic hearing loss is associated with a mutation in the SLC26A4 gene. In another aspect, the present disclosure provides a polynucleotide, vector, rAAV, cell, or composition described herein for use in a method for treating a pendrin-associated disease (e.g., Pendred syndrome or autosomal recessive nonsyndromic deafness) in a subject in need thereof.

[0172] In another aspect, the present disclosure provides a polynucleotide, vector, rAAV, cell, or composition described herein for use in the manufacture of a medicament for specifically expressing pendrin in cells that normally express the SLC26A4 gene in a subject. In another aspect, the present disclosure provides a polynucleotide, vector, rAAV, cell, or composition described herein for use in the manufacture of a medicament for treating genetic hearing loss in a subject in need thereof. In some embodiments, the genetic hearing loss is associated with a mutation in the SLC26A4 gene. In another aspect, the present disclosure provides a polynucleotide, vector, rAAV, cell, or composition described herein for use in the manufacture of a medicament for treating a pendrin-associated disease (e.g., Pendred syndrome or autosomal recessive nonsyndromic deafness) in a subject in need thereof.VI. AAV packaging systems, kits, and related compositions

[0173] In one aspect, the present disclosure provides packaging systems for recombinant preparation of a recombinant adeno-associated virus (rAAV) described herein. Such packaging systems generally comprise a first nucleotide encoding one or more AAV Rep proteins; a second nucleotide encoding a capsid protein of any of the AAVs described herein; and a third nucleotide sequence comprising any of the rAAV vectors described herein, wherein the packaging system is operative in a cell for enclosing the rAAV vector in the capsid to form the rAAV.

[0174] In some embodiments, the packaging system comprises a first vector comprising the first nucleotide sequence encoding the one or more AAV Rep proteins and the second nucleotide sequence encoding the AAV capsid protein, and a second vector comprising the third nucleotide sequence comprising the rAAV vector. As used in the context of a packaging system as described herein, a “vector” refers to a nucleic acid molecule that is a vehicle for introducing nucleic acids into a cell (e.g., a plasmid, a virus, a cosmid, an artificial chromosome, etc.).

[0175] Any AAV Rep protein can be employed in the packaging systems disclosed herein. In some embodiments of the packaging system, the Rep nucleotide sequence encodes an AAV2 Rep protein. Suitable AAV2 Rep proteins include, without limitation, Rep 78 / 68 or Rep 68 / 52.

[0176] In some embodiments, the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes. In some embodiments, the packaging system further comprises a third vector, e.g, a helper virus vector, comprising the fourth nucleotide sequence comprising the one or more helper vims genes. The third vector may be an independent third vector, part of the first vector, or part of the second vector.

[0177] In some embodiments of the packaging system, the helper virus is selected from the group consisting of adenovirus, herpes vims (including herpes simplex vims (HSV)). poxvirus (such as vaccinia vims), cytomegalovirus (CMV), and baculovirus. In some embodiments of the packaging system, where the helper vims is adenovirus, the adenovirus genome comprises one or more adenovirus RNA genes selected from the group consisting of El, E2, E4 and VA. In some embodiments of the packaging system, where the helper virus is HSV, the HSV genome comprises one or more of HSV genes selected from the group consisting of UL5 / 8 / 52, ICPO, ICP4, ICP22 and UL30 / UL42.

[0178] In some embodiments of the packaging system, the first, second, and / or third vector are contained within one or more plasmids. In some embodiments, the first vector and the third vector are contained within a first plasmid. In some embodiments the second vector and the third vector are contained within a second plasmid.

[0179] In some embodiments of the packaging system, the first, second, and / or third vector are contained within one or more recombinant helper viruses. In some embodiments, the first vector and the third vector are contained within a recombinant helper vims. In some embodiments, the second vector and the third vector are contained within a recombinant helper virus.

[0180] In a further aspect, the present disclosure provides a method for recombinant preparation of an rAAV described herein, wherein the method comprises transfecting or transducing a cell with a packaging system as described herein under conditions operative for enclosing the rAAV vector in the capsid to form the rAAV as described herein. Exemplary methods for recombinant preparation of an rAAV include transient transfection (e.g., with one or more transfection plasmids containing a first, and a second, and optionally a third vector as described herein), viral infection (e.g, with one or more recombinant helper viruses, such as a adenovirus, poxvirus (such as vaccinia vims), herpes virus (including HSV, cytomegalovims, or baculovirus), containing a first, and a second, and optionally a third vector as described herein), and stable producer cell line transfection or infection (e.g., with a stable producer cell, such as a mammalian or insect cell, containing a Rep nucleotide sequence encoding one or more AAV Rep proteins and / or a Cap nucleotide sequence encoding one or more AAV capsidproteins as described herein, and with an rAAV vector as described herein being delivered in the form of a plasmid or a recombinant helper virus).

[0181] In another aspect, the present disclosure provides kits for use of the compositions described herein, e.g., in therapeutic or research applications. A kit may include one or more containers housing components (e g., a polynucleotide or rAAV described herein) and instructions for use. In some embodiments, the kit comprises one or more components described herein and instructions describing the intended application and the proper use of said components. In some embodiments, components in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the components. Kits for research purposes may contain the components in appropriate concentrations or quantities for performing various experiments.

[0182] In some embodiments, the present disclosure provides a kit for administering an rAAV as described herein. In some embodiments, the kit comprises a container housing the rAAV, and devices (e.g, syringes) for extracting the rAAV from the housing. In some embodiments, the device for extracting the rAAV from the housing is also used for administration (e.g, via injection).

[0183] In some embodiments, the present disclosure provides a kit for producing an rAAV (e.g, according to the methods provided herein), the kit comprising a container housing a polynucleotide comprising a transgene encoding a protein (e.g., pendrin). In some embodiments, the kit further comprises a container housing a polynucleotide encoding an AAV capsid protein, e.g, an AAV-S capsid protein. In some embodiments, the kit further comprises vectors comprising the rep / cap genes and / or the host for producing the rAAV.

[0184] In some embodiments, a kit provided here is designed to facilitate use of the methods described herein, e.g, by researchers and / or clinicians, and can take many different forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g, in solution), or in solid form (e.g, a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g, to an active form), for example, by the addition of a suitable solvent or other medium (for example, water or a cell culture medium), which may or may not be provided in the kit. As used herein, '■instructions" can include a component of instruction and / or promotion, and typically involve written instructions on or associated with the packaging. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, CD-ROM, website links for downloadable file, etc.), Internet, and / or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.

[0185] A kit provided herein may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and administering it to a subject. The kit may include a container housing the rAAV described herein. The rAAV may be in the form of a liquid, gel, or solid (powder). The rAAV may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, the rAAV may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively, the kit may include the rAAV premixed and shipped in a syringe, vial, tube, or other container.

[0186] Exemplar^' embodiments of the invention will be described in more detail by the following examples. These examples are offered by way of illustration, and not by way of limitation.EXAMPLESExample 1. Vectors used in Examples 2-5.

[0187] Various exemplary vectors were designed, packaged into an AAV-S capsid, and characterized as detailed in Examples 2-5. The SLC26A4-UTR vector, used in Examples 2 and 5, comprises, from 5' to 3', a 5' ITR, a CBA promoter, a 5' UTR, a human pendrin protein coding sequence, a 3' UTR, a bovine growth hormone (BGH) polyadenylation sequence, and a 3' ITR (FIG. 2, top panel). The SLC26A4-HA-UTR vector, used in Examples 3, 4. and 6, comprises, from 5' to 3', a 5' ITR, a CBA promoter, a 5' UTR, a human pen drin protein coding sequence, an HA tag sequence, a 3' UTR, a bovine growth hormone polyadenylation sequence, and a 3' ITR (FIG. 2, middle panel). The SL26A4-HA vector, used in Example 4, comprises, from 5' to 3', a 5' ITR, a CBA promoter, a human pendrin protein coding sequence, an HA tag, a bovine growth hormone polyadenylation sequence, and a 3' ITR (FIG. 2, bottom panel). The sequences of these genetic elements are set forth in Table 1.Table 1. Selected genetic elements in pendrin expression vectors SLC26A4-UTR, SLC26A4- HA-UTR, and SLC26A4-HA.Example 2. Development of an in vitro assay for measurement of pendrin activity.

[0188] This Example describes development and characterization of a method to examine the in vitro activity of a viral vector encoding pendrin (e.g., as provided herein), as well as use of the method to verify expression of functional pendrin protein from an exemplary' viral vector (SLC26A4-UTR) capable of anion exchange in a dose-dependent manner in vitro.

[0189] Development of a potency assay for adeno-associated virus vectors (AAV) to measure the relevant biological activity or properties and claimed mechanism of action is a key process development step in defining quality' control for any clinical AAV therapeutic. According to FDA guidelines, potency assays should ideally be quantitative and should correlate with the clinical response - robust potency assays are fundamental in showing comparability between vector lots, AAV production process development, and vector stability studies. They also sen e as an important quality control measure to ensure that only vectors that meet pre-defmed criteria are used during clinical investigation or released following market approval (U.S. Department of Health and Human Services. Food and Drug Administration. Guidance for Industry. Potency Tests for Cellular and Gene Therapy Products (2011)).

[0190] Pendrin is a 780 amino acid membrane anion exchange protein encoded by the SLC26A4 gene. Mutations in SLC26A4 account for one of the most common forms of inherited hearing loss. In the inner ear, pendrin is thought to mediate chloride and bicarbonate anion exchange to maintain proper endolymphatic pH and thereby support formation of the endocochlear potential (Wangemann et al., 2006, Am. J. Physio. Renal Physiol. 292:F1345- F1353). Pendrin’s anion exchange activity facilitated the development of an in vitro fluorometric assay exploiting the fluorescence sensitivity of yellow fluorescent protein (YFP) to anion-dependent fluorescence quenching (Dossenaef al., 2006, Cell Physiol. Biochem. 18(1- 3):67-74; Matulevicius, et al., 2022, J. Clin. Med. 1 1(19):5549). This assay was adapted to show that the SLC26A4-UTR vector produces a functional human pendrin protein in vitro,thereby establishing the basis for the development of an in vitro, quantitative potency assay measuring the biological activity of pendrin.

[0191] A schematic representation of the pendrin activity assay is show n in FIG. 1. Briefly, AAV was thawed on ice and used to transduce HEK293 Phoenix Eco cells at different MOIs. Transduced cells were then transfected with a plasmid encoding an anion-sensitive YFP mutant. Three replicates for each MOI were assessed for anion-induced YFP fluorescence quenching. A schematic representation of the single-stranded AAV vector SLC26A4-UTR used in the study is shown in FIG. 2, top panel.Materials and methods

[0192] SLC26A4-UTR vector was produced using a standard adherent HEK293 triple transfection system. AAV vector was purified using cesium chloride density gradient ultracentrifugation and formulated in a standard formulation buffer of IX PBS containing 0.001% Pluronic F-68. Vectors were concentrated using an Amicon Ultra-2 centrifugal filter unit with a 100 kDa MWCO and adjusted to the desired titer in formulation buffer. All vectors met internal QC standards for titer, purity, full / empty capsid >80%, endotoxin <lEU / mL, in vitro expression, vector genome identity, and aggregation.

[0193] One day pre-transduction, HEK293 Phoenix Eco cells (ATCC, CRL-3214) were plated on black-walled clear-bottom 96-well plates (Costar, 3603) coated with poly-L-lysine (Sigma, P4707). Cells were then transduced with SLC26A4-UTR at select multiplicity of infection (MOI) values, normalizing for amount of total vector and formulation buffer between samples. 24 hours post-transduction, cells were transfected with an anion-sensitive mutant of YFP (YFP H148Q / I152L) using Lipofectamine 3000 (Thermo Fisher, L3000001). YFP fluorescence quenching was quantified at 72 hours post-transduction using a Biotek Synergy HT plate reader (Excitation 485nm, center wavelength (CWL) bandwidth 20nm; Emission 535nm. CWL bandwidth 20nm; Gain 80). Specifically, cells were bathed in 70 pl of a high Cl’ solution (in mM: KC1 2, NaCl 135, CaC12 1 , MgC12 1 , D-glucose 10, 4-(2-hy droxy ethyl)- 1 - piperazineethane sulfonic acid (HEPES) 20) and baseline YFP fluorescence was quantified every 4 seconds for 8 seconds (3 total readings). Then, 140 pl of a high I’ solution (in mM: KC1 2, Nal 135, CaC12 1, MgC12 1, D-glucose 10, HEPES 20) was added to the wells and YFP fluorescence was quantified every 4 seconds for 2.5 minutes. To test reversibility of the assay, assay buffers were removed post-YFP fluorescence quenching quantification and replaced with normal cell growth media. YFP fluorescence quenching assay was then repeated as described above.

[0194] After measuring YFP fluorescence quenching, transduced cells w ere fixed w ith 4% paraformaldehyde for 12 minutes, washed with PBS and then blocked overnight at 4°C with blocking buffer (PBS + 1% BSA + 2% Goat serum + 0.2% Triton). The next day, blocking buffer was removed and cells were stained with a mouse anti-pendrin primary7antibody (Santa Cruz, sc-518130) diluted 1:50 in blocking buffer followed by a goat anti-mouse 800 secondary antibody (Azure Biosy stems, AC2135) diluted 1 :250 in blocking buffer. Stained wells were imaged on a Li-Cor Odyssey CLx.Results and discussion

[0195] As shown in FIG. 3, the YFP fluorescence quenching assay described above is capable of measuring pendrin-mediated anion exchange in a dose-dependent manner, and the SLC26A4-UTR vector produces a functional pendrin protein capable of dose-dependent anion exchange. Cells transduced with SLC26A4-UTR at increasing MOIs showed dose-dependent decreases in YFP fluorescence upon addition of the high I’ solution relative to control cells transduced with formulation buffer (FIG. 3, top panel), which indicates increased YFP quenching as a result of pendrin-mediated anion exchange. After measuring pendrin activity, dose-dependent expression of pendrin in cells transduced with the SLC26A4-UTR vector was verified using immunofluorescence with an anti-pendrin antibody (FIG. 3, bottom panel).

[0196] YFP fluorescence quenching by pendrin-mediated anion exchange was also shown to be reversible. As described above, cells transduced with the SB-008 lc9 vector show a dosedependent decrease in YFP fluorescence upon addition of the high I' solution (FIG. 4, from first ‘'Iodide addition” to ‘'Media change”). After reading YFP fluorescence, the cells were returned to growth media (FIG. 4, ‘Media change”) and incubated for 2 min at room temperature prior to running the pendrin activity7as described a second time (FIG. 4, “Chloride addition” and second “Iodide addition”), which showed similar dose-dependent pendrin activity (FIG. 4, after second “Iodide addition”).

[0197] This Example demonstrates development of an assay capable of measuring the anion exchange activity7of pendrin after transduction w ith the SB-008 lc9 vector in a significant, dose-dependent, and reversible manner over a 2-log MOI range. Additionally, the assay was sensitive enough to detect significant differences in pendrin activity with 0.5-log differences in MOI. The development of this assay lays the foundation for the future development of an in vitro potency assay7for additional viral vectors encoding pendrin (e.g., AAV-S vectors encoding pendrin, e.g, as described herein).Example 3. Targeted expression of pendrin in mouse cochlea cells.

[0198] This Example describes characterization of an AAV vector, SLC26A4-HA-UTR, designed for expression of pendrin in inner ear cells that have endogenous pendrin expression. AAV-mediated pendrin expression in mouse (Mus musculus) cochlea was analyzed, and the expression pattern was compared to that of endogenous pendrin expression. The results show that the SLC26A4-HA-UTR vector drives pendrin expression in the targeted cells, including root cells, spindle cells, epithelial cells of the spiral prominence, and outer sulcus cells. In addition, the results show that expression is stronger at the higher dose and increases regardless of dose at the latest timepoint tested.

[0199] Congenital hearing loss affects about 1 in 500 newborns and most of the cases are genetic in cause (Morton and Nance, 2006, New England Journal of Medicine 354:2151-2164). SLC26A4 is among the top four genes that are associated with hearing loss (Sloan-Heggen el al.. 2016, Hum. Genet. 135:441-450). Mutations in the gene cause Pendred syndrome (PDS) and autosomal recessive non-syndromic deafness 4 (DFNB4). Both disorders are autosomal recessive, making them good candidates for replacement gene therapy. SLC26A4 encodes pendrin, which promotes transmembrane exchange of anions (such as Cl" and I") and bases (such as HCO3 and OH ) across plasma membranes of epithelial cells (Alper and Sharma, 2013, Mol. Aspects Med. 34:494-515). In the mouse inner ear, pendrin is expressed in mitochondria-rich cells of the endolymphatic sac, transitional cells of the vestibule, outer sulcus cells, root cells and spindle cells in the cochlea (Everett et al., 1999, Proc. Nat. Acad. Sci. 96:9727-9732: Everett et al., 2001, Hum. Mol. Genet. 10: 153-161; Rovaux et al., 2003, Journal of the Association for Research in Otolaryngology 4:394-404; Li et al., 2013, PLoS Genet. 9:el003641). Pendrin expression in these cells is required to keep the ion balance in the inner ear and is thus necessary for hearing (Li et al., 2013, PLoS Genet. 9:el003641; Wangemann et al., 2006, Am. J. Physio. Renal Physiol. 292:F1345-F1353).

[0200] Vectors were designed to express pendrin in a way that mimics endogenous protein expression in order to rescue hearing deficits caused by SLC26A4 mutations. To achieve this, the SLC26A4-HA-UTR vector was designed with upstream and downstream regulatory elements of the human SLC26A4 gene. After local injection of the SLC26A4-HA-UTR vector, expression of pendrin driven by the AAV was analyzed in mouse ears and compared to endogenous pendrin expression.

[0201] A schematic representation of the single-stranded AAV vector SLC26A4-HA-UTR used in the study is shown in FIG. 2, middle panel.

[0202] The design of the study is shown in Table 2, below. Briefly, the SLC26A4-HA-UTR vector was injected at 2 different doses into mice, and pendrin expression was evaluated after 5 days or 18 days.Table 2. Study design.Materials and methods

[0203] Vectors were produced using a standard adherent HEK293 triple transfection system and purified using cesium chloride density gradient ultracentrifugation to achieve the desired titer. All vectors were formulated in formulation buffer containing IX PBS+ 0.001% Pluronic F-68. Vectors were concentrated using MilhporeSigma Amicon ultrafiltration devices to achieve desired titers. All vectors met internal QC standards for purity, full / empty capsid >80%, endotoxin <lEU / mL, infectivity, vector genome identity, and aggregation. Vector titers were determined by ddPCR in a Bio-Rad QX200 using probes and primers to the bGH polyadenylation signal sequence.

[0204] Wild-type C57BL / 6J mice were obtained from the Jackson Laboratory. Animal handling, breeding, and all procedures were performed in compliance with NIH ethics guidelines and with a protocol approved by the Animal Care Committee of Harvard Medical School.

[0205] P1-P2 pups were anesthetized, and round window membrane (RWM) injection surgeries were performed as previously described (G orgy et al., 2017, Molecular Therapy 25:379-391; Ivanchenko et al., 2021, Molecular Therapy-Methods & Clinical Development 21 :382-398).

[0206] Cryopreservation and sectioning were performed as previously described (Ivanchenko et al., 2021, Molecular Therapy-Methods & Clinical Development 21:382-398). Cryosections were brought to room temperature and washed 2X with HBSS for 10 minutes. Sections were then blocked in HBSS containing 10% donkey serum and 0.3% Triton X-100 for 1 hour at room temperature (RT). Primary' antibodies, guinea pig anti-parvalbumin (Synaptic Systems, Cat.#195 004, 1 : 100), rabbit anti-HA antibodies (Cell Signaling, #3724 1:200), and goat anti- ANAX1 antibodies (LS-B3028-50, 1: 100), were diluted in HBSS containing 10% goat serum and 0.1 % Triton X-100, and incubated at RT for 24 hours in a wet chamber. After removing the primary antibody, slides were washed with HBSS for 3 x 10 min and then blocked for 30 min in HBSS with 10% donkey serum. Secondary antibodies, donkey anti-guinea pig (Jackson ImmunoResearch, 706-605-148, 1:500)1 donkey anti-rabbit (Thermo Fisher Scientific, A32790, 1:500); donkey anti-goat (Thermo Fisher Scientific, A32849, 1:500), as well as Hoechst (Thermo Fisher Scientific, H3570, 1:2000) or DAPI (Sigma, D9542, 1:500), and Phalloidin (Thermo Fisher Scientific, A12381, 1 :200) were diluted in HBSS with 10% donkey serum and incubated at RT overnight in a wet chamber. The sections were then washed with HBSS 3 x 10 min before mounting with ProLong Gold Antifade mounting medium (Thermo Fisher Scientific). The slides were left to dry completely in the dark for 24 to 48 hours before imaging.

[0207] Images were captured with Leica DMi8 system. Acquisition settings were kept the same for all samples among all groups.Results and discussion

[0208] In the mouse cochlea, pendrin is expressed in outer sulcus cells, root cells, spindle cells, and epithelial cells of the spiral prominence (Everett et al., 1999, Proc. Nat. Acad. Sci. 96:9727-9732; Everett e / al., 2001. Hum. Mol. Genet. 10: 153-161; Rovaux etal., 2003, Journal of the Association for Research in Otolaryngology 4:394-404; Li et al., 2013, PLoS Genet. 9:el 003641). To recapitulate endogenous pendrin expression, the AAV vector SLC26A4-HA- UTR (FIG. 2, middle panel) was designed to encode human pendrin under the control of a ubiquitous chicken (Lactin (CBA) promoter with the 5' untranslated region (5' UTR) and an engineered version of the 3' UTR from the human SLC26A4 gene. Since antibodies cannot distinguish between mouse and human pendrin, a human influenza hemagglutinin (HA) tag was included for detection of exogenous pendrin protein using anti-HA antibodies.

[0209] To study AAV -mediated pendrin expression, AAV was injected into the mouse cochlea through the round window membrane (RWM) at either 1E10 vg / ear or 1E11 vg / ear.The cochleae were harvested and cryosectioned either 5 days post injection (dpi) or 18 dpi. Exogenous pendrin expression in the mid-modiolar sections was examined using anti-HA antibodies, and uninjected cochleae were used as negative controls. Exogenous pendrin expression was detected at both doses and at both time points. At both time points, exogenous pendrin expression was detected in more cells at the higher dose (1E11 vg / ear) than at the lower dose (1E10 vg / ear). At the same dose, longer expression time (18 dpi) led to broader exogenous pendrin expression compared with 5 dpi. Exogenous pendrin expression was similar in all three turns. In addition, exogenous pendrin expression was detected in all cell types that express endogenous pendrin.

[0210] This study demonstrates that the SLC26A4-HA-UTR vector expresses human pendrin in the cell types that express endogenous pendrin in mice, and that the expression is time and dose dependent.Example 4. Effect of UTRs on targeted expression of pendrin in mouse cochlea cells.

[0211] This Example describes further characterization of the SLC26A4-HA-UTR vector, designed using the 5' UTR and 3' UTR of the human SLC26A4 gene to express pendrin in cochlear cells that have endogenous pendrin expression. In this study, AAV expression in mouse (Mus musculus) cochlea was examined, and the expression pattern was compared to vectors without the UTRs. The results show that the UTRs play a role in restricting AAV- mediated gene expression to the cells that have endogenous pendrin expression in the mouse cochlea.

[0212] Schematic representations of the single-stranded AAV vectors SLC26A4-HA-UTR and SLC26A4-HA used in the study are shown in FIG. 2, middle and bottom panels, respectively.

[0213] The design of the study is show n in Table 3, below. Briefly, the SLC26A4-HA-UTR and SLC26A4-HA vectors were injected into mice, and pendrin expression was evaluated after 18 days.Table 3. Study design.Materials and methods

[0214] Vectors were produced using a standard adherent HEK293 triple transfection system and purified using cesium chloride density gradient ultracentrifugation to achieve the desired titer. All vectors were formulated in formulation buffer containing IX PBS+ 0.001% Pluronic F-68. Vectors were concentrated using MilliporeSigma Amicon ultrafiltration devices to achieve desired titers. All vectors met internal QC standards for purity, full / empty capsid >80%, endotoxin <lEU / mL, infectivity', vector genome identity, and aggregation. Vector titers were determined by ddPCR in a Bio-Rad QX200 using probes and primers to the bGH polyadenylation signal sequence.

[0215] Wild-type C57BL / 6J mice were obtained from the Jackson Laboratory. Animal handling, breeding, and all procedures were performed in compliance with NIH ethics guidelines and with a protocol approved by the Animal Care Committee of Harvard Medical School.

[0216] P1-P2 pups were anesthetized, and round window membrane (RWM) injection surgeries were performed as previously described (G orgy et al., 2017, Molecular Therapy 25:379-391; Ivanchenko et al., 2021, Molecular Therapy-Methods & Clinical Development 21 :382-398).

[0217] Cry opreservation and sectioning were performed as previously described (Ivanchenko et al., 2021, Molecular TherapyMethods & Clinical Development 21 382-398).

[0218] Cryosections were brought to room temperature and washed 2X with HBSS for 10 minutes. Sections were then blocked in HBSS containing 10% donkey serum and 0.3% Triton X-100 for 1 hour at room temperature (RT). Primary antibodies, guinea pig anti-parvalbumin (Synaptic Systems, Cat.#195 004, 1 : 100), rabbit anti-HA antibodies (Cell Signaling, #3724 1:200), and goat anti-ANAXl antibodies (LS-B3028-50, 1: 100), were diluted in HBSS containing 10% goat serum and 0.1% Triton X-100, and incubated at RT for 24 hours in a wet chamber. After removing the primary antibody, slides were washed with HBSS for 3 x 10 min and then blocked for 30 min in HBSS with 10% donkey serum. Secondary antibodies, donkey anti-guinea pig (Jackson ImmunoResearch, 706-605-148, 1 :500); donkey anti-rabbit (Thermo Fisher Scientific, A32790, 1 :500); donkey anti-goat (Thermo Fisher Scientific, A32849, 1:500), as well as Hoechst (Thermo Fisher Scientific, H3570, 1:2000) or DAPI (Sigma, D9542, 1:500), and Phalloidin (Thermo Fisher Scientific, A12381, 1:200) were diluted in HBSS with 10% donkey serum and incubated at RT overnight in a wet chamber. The sections were then washed with HBSS 3 x 10 min before mounting with ProLong Gold Antifade mounting medium(Thermo Fisher Scientific). The slides were left to dry completely in the dark for 24 to 48 hours before imaging.

[0219] Images were captured with Leica DMi8 system. Acquisition settings were kept the same for all samples among all groups.Results and discussion

[0220] In the mouse cochlea, pendrin is expressed in outer sulcus cells, root cells, spindle cells, and epithelial cells of the spiral prominence (Everett et al.. 1999, Proc. Nat. Acad. Sci. 96:9727-9732; Everett etal., 2001, Hum. Mol. Genet. 10: 153-161; Rovaux etu / ., 2003, Journal of the Association for Research in Otolaryngology’ 4:394-404; Li et al., 2013, PLoS Genet. 9:el003641). To recapitulate the endogenous pendrin expression, the AAV vector SLC26A4- HA-UTR (FIG. 2, middle panel) was designed to encode human pendrin under the control of a ubiquitous chicken (3-actin (CBA) promoter with the 5' untranslated region (5' UTR) and an engineered version of the 3' UTR from the human SLC26A4 gene to restrict AAV-mediated expression. In this study, the effect of the UTRs was examined by comparing pendrin expression from the SLC26A4-HA-UTR vector to expression from the SLC26A4-HA vector. Since antibodies cannot distinguish between mouse and human pendrin, a human influenza hemagglutinin (HA) tag was included for detection of exogenous pendrin protein using anti- HA antibodies.

[0221] To study the AAV-mediated exogenous pendrin expression, AAVs with both vectors were separately injected into the mouse cochlea through the round window membrane (RWM) at 1E10 vg / ear. The cochleae were harvested and cryosectioned at 18 days post injection (dpi). Exogenous protein expression in the mid-modiolar sections was examined using anti-HA antibodies, and uninjected cochleae were used as negative controls. Without UTRs, exogenous pendrin expression was detected throughout the cochlea. With UTRs. exogenous pendrin expression was more restricted and mimicked endogenous pendrin expression. Exogenous pendrin expression was similar in all three turns.

[0222] This study demonstrates that the UTRs increase the specificity of AAV-mediated exogenous pendrin expression to the cells that express endogenous pendrin in mice.Example 5. Exogenous pendrin expression in mouse models of genetic hearing loss.

[0223] This Example describes use of an AAV vector, SLC26A4-UTR, to express exogenous pendrin in the inner ear of SLC26A4 mutant mice. The results show that exogenous pendrinexpression preserved both hearing thresholds, as measured by auditory brainstem responses (ABR). and outer hair cell (OHC) development in the mutant mice.

[0224] Many mouse models have been made to study the pathology of PDS and DFNB4, but these models either have normal hearing or are profoundly deaf, unlike the less severe human phenoty pes. The only exception is the L236P knock-in mouse model, which has variable hearing loss (Wen et al., 2019, Biochem. Biophys. Res. Commun. 515:359-365), making it a good model for DFNB4.

[0225] The SLC26A4-UTR vector was designed with upstream (5') and downstream (3') regulatory elements (UTRs) of the human SLC26A4 gene and injected into the inner ear of the SLC26A4 L236P mice. Compared with uninjected ears, ears injected with AAV overexpressing pendrin have preserved hearing, as shown by acoustic brainstem response (ABR), as well as preserved OHC development.

[0226] A schematic representation of the single-stranded AAV SLC26A4-UTR vector used in the study is shown in FIG. 2, top panel. The vector was injected into mutant mice at varying doses, and ABR was recorded in the injected mice and in negative control (uninjected) mice (n=129), followed by histological examination.Materials and methods

[0227] Vectors were produced using a standard adherent HEK293 triple transfection system and purified using cesium chloride density' gradient ultracentrifugation to achieve the desired titer. All vectors were formulated in formulation buffer containing IX PBS+ 0.001% Pluronic F-68. Vectors were concentrated using MilliporeSigma Amicon ultrafiltration devices to achieve desired titers. All vectors met internal QC standards for purity, full / emply capsid >80%, endotoxin <lEU / mL, infectivity, vector genome identity, and aggregation. Vector titers were determined by ddPCR in a Bio-Rad QX200 using probes and primers to the bGH polyadenylation signal sequence.

[0228] SLC26A4 L236P mice were obtained from Shandong University (Wen et al., 2019, Biochem. Biophys. Res. Commun. 515:359-365) and backcrossed to CBA / CaJ mice for one generation. Animal handling, breeding, and all procedures w ere performed in compliance w ith NIH ethics guidelines and with a protocol approved by the Animal Care Committee of Harvard Medical School.

[0229] P1-P2 pups were anesthetized, and round yvindow membrane (RWM) injection surgeries w ere performed as previously described (Gy orgy et al., 2017, Molecular Therapy 25:379-391; Ivanchenko et al., 2021 , Molecular Therapy-Methods & Clinical Development21 :382-398). Briefly, a small incision was made underneath the left external ear. The incision was enlarged, and soft tissues were pushed apart to expose the bulla. The round window niche was localized visually. The viral vector solution was injected with a micropipette needle at a rate of lOO nL / min using a Nanoliter 2000 Injector (World Precision Instruments). A total of 1 pL of test article was administered to the left ear. The surgical incision was closed using a 7-0 Vicryl surgical suture. Standard postoperative care was applied after the injection.

[0230] Hearing assessments were carried out via auditory brainstem response (ABR) testing at P30 as described in Peters et al., 2023, Molecular Therapy 31 (8):2439-2453. Briefly, ABRs were recorded using a custom EPL acoustic system (Massachusetts Eye and Ear, Boston, MA, USA) in a custom-made acoustically and electrically shielded chamber (Eckel Industries). Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg)-xylazine (10 mg / kg) cocktail and placed on a temperature-controlled heating pad set to 37° C for the duration of the experiment. Acoustic stimuli were delivered to the left ears of animals via a custom acoustic assembly consisting of two electrostatic drivers as sound sources and a miniature microphone at the end of a probe tube to measure sound pressure in situ. ABRs were recorded using three subdermal needle electrodes: active electrode in the scalp between the ears, reference electrode just below the pinna, and ground electrode in the back near the tail.

[0231] 5-ms tone-pip stimuli with a 0.5 ms rise-fall time at frequencies from 5.6-32 kHz were delivered in alternating polarity. The response was amplified (xlO.OOO), band-pass filtered (0.3-3 kHz), and averaged (x512) with a PC-based data acquisition system using the Cochlear Function Test Suite software package (Massachusetts Eye and Ear, Boston, MA, USA). Sound levels were incremented in 5-dB steps, from ~20 dB below threshold up to 125 dB sound pressure level (SPL). A custom-made ABR peak analysis software was used to manually select and record the ABR thresholds. ABR thresholds were confirmed by visual examination as the lowest stimulus level where decreasing peak amplitude and increasing peak latency could be confirmed.

[0232] Cryopreservation and sectioning were performed as previously described. Briefly, cryosections were brought to room temperature and washed 2 times with HBSS for 10 minutes. Sections were then blocked in HBSS containing 10% donkey serum and 0.3% Triton X-100 for 1 hour at room temperature (RT). Primary antibodies: Pendrin (Slc26a4) [1:50] (BiCell Scientific, Catalog #20501, Rabbit Polyclonal); or Myosin7a [1: 100] (Proteus, Catalog #25- 6790, Rabbit Polyclonal) were diluted in HBSS containing 10% donkey serum and 0.1% Triton X-100 and incubated at RT for 24 hours in a wet chamber. After removing the primary antibody, slides were washed with HBSS 3 times for 10 minutes and then blocked for 30minutes in HBSS with 10% donkey serum. Secondary antibodies, Donkey-anti-Rabbit 488 [1 :500] (Thermo-Fisher (Invitrogen), Catalog #A32790), and 50% Diluted Hoechst 33342 [1 : 1000] (Thermo-Fisher, Catalog #H3570) were diluted in HBSS with 10% donkey serum and incubated at RT overnight in a wet chamber away from light. The sections were then washed with HBSS 3 times for 10 minutes before mounting with ProLong Gold Antifade mounting medium (Thermo Fisher Scientific). The slides were left to dry completely in the dark for 24 to 48 hours before imaging. Images were captured with a Leica DMi8 microscope at 40X.Results and discussion

[0233] To express exogenous pendrin in cells that have endogenous pendrin expression, the SLC26A4-UTR vector was designed to encode human pendrin under the control of a ubiquitous CBA (chicken [3-actin) promoter and the 5' UTR and an engineered 3' UTR from the human SLC26A4 gene were included to restrict the AAV expression. AAV with the SLC26A4-UTR vector were injected into the inner ears of SLC26A4 L236P homozygous (HOM) mice at P1-P2, through round window membrane (RWM), and ABRs were recorded around 30 days post-injection (FIG. 5). Mice with the knock-in L236P mutation exhibit variable hearing loss ranging from normal to profound. Considering the variability of hearing thresholds in the L236P mouse model, 30-60 animals per group were included to power the study. Treated ears dosed at the mid dose, high dose, and higher dose levels showed vary ing preservation of ABR thresholds at p30, and some animals had thresholds in the normal hearing range. Treatment of ears at the low dose did not have a noticeable effect on ABR thresholds (FIG. 5)

[0234] After ABR thresholds were recorded around P30, cochleae from uninjected L236P HOM mice and from L236P HOM mice treated with various doses of AAV-SLC26A4-UTR were harvested and processed for cryosections, and inner hair cells (IHCs) and OHCs were examined. The inner hair cells remained intact in the uninjected L236P HOM mice, but the outer hair cells (OHCs) were mostly missing. In the AAV-SLC26A4-UTR treated cochlea treated with mid dose or higher, the OHCs were mostly present. At low dose, about half of the analyzed cochleae had OHCs. These findings were consistent with the ABR results, where AAV-SLC26A4-UTR at low dose had no effect on ABR thresholds, while AAV-SLC26A4- UTR at mid dose or higher preserved hearing function (FIG. 5).

[0235] These studies demonstrate that the SLC26A4-UTR vector can preserve hearing and OHCs in a mouse model for DFNB4, SLC26A4 L236P.Example 6. Exogenous pendrin expression in non-human primates.

[0236] This Example describes use of an AAV vector, SLC26A4-HA-UTR, to express exogenous pendrin in the inner ear of non-human primate (NHP) cynomolgus macaques. The results show that the SLC26A4-HA-UTR AAV vector is able to express pendrin in DFNB4- relevant cell types in the NHP inner ear. A schematic representation of the single-stranded AAV SLC26A4-HA-UTR vector used in the study is show n in FIG. 2, middle panel. Because endogenous pendrin protein is expressed in the primate inner ear (Hosoya et al., 2016, Neurosci. Res. 110: 1-10), an HA-tagged version of AAV-SLC26A4-HA-UTR was used to allow differentiation between vector-derived pendrin protein and endogenous protein. NHP ears were injected with the vector at vary ing doses, and histological examination was performed five weeks later.Materials and Methods

[0237] Vectors were produced as described above.

[0238] All in-life procedures were performed according to animal use guidelines and approved procedures. Animal enrichment, environmental conditions, food, and water were provided according to standard practices.

[0239] 20 pL of vector was delivered through the round w indow membrane (RWM) to each ear with animals under anesthesia. After retroauricular incision, the mastoid bone was exposed and cortical mastoidectomy was performed. When the fossa incudis was reached, a 1- to 2 -mm facial recess was identified. A low' drill speed and continuous fluid irrigation was used when opening the facial recess to approach the RWM. Under direct visualization through the operating microscope, the needle was manually positioned into the round window and the needle held in place during the infusion. Viral vector injection was performed using a microinjection syringe pump (World Precision Instruments) at 2.0 pL / min for 10 min, using a Hamilton syringe connected via a thin, relatively non-compliant silicone tube to a 29G, approximately 2-cm-long, sharp-end stainless steel injection needle. The syringe and catheter were filled with sterile saline and the needle backfilled with 30 pL of vector separated from saline by an air bubble in the catheter. Total procedure duration for each animal was approximately 5-6 hours.

[0240] Cryopreservation and sectioning were performed as described (Ivanchenko el al., 2021, Molecular Therapy -Methods & Clinical Development 21 :382-398). Briefly, cryosections were brought to room temperature and washed IX with HBSS for 5 minutes. Sections werethen blocked in HBSS containing 10% donkey serum and 0.1% Triton X-100 for 2 hours at room temperature (RT). Primary antibodies, rabbit anti-HA (Cell Signaling, #C29F4, 1 :200); mouse anti-Myo7A (Santa Cruz, 1:50) were diluted in HBSS with 10% donkey serum, and incubated at RT overnight. After removing the primary antibody, slides were washed with HBSS for 3 x 10 min and then blocked for 1 hour in HBSS with 10% donkey serum. Secondary antibodies, donkey anti-rabbit Alexa 488 and donkey anti-mouse Alexa 568 were diluted in HBSS with 10% donkey serum and incubated at RT overnight. The next day, the sections were washed twice with HBSS for 10 min each, and DAP1 (2 mg / ml) was used to counter stain the nuclei for 8 min. The sections were then washed with HBSS 2 x 10 min before mounting with ProLong Gold Antifade Mountant (Fisher). The sections were left to dry completely before imaging.Results and discussion

[0241] As noted above, the SLC26A4-HA-UTR vector was designed to express exogenous pendrin in cells that have endogenous pendrin expression. AAV with the SLC26A4-HA-UTR vector were injected into six ears from three NHP cynomolgus macaques at low and high doses via trans-mastoid surgery into the RWM. Five weeks after the surgery, the temporal bones were collected and processed for histology and imaging. Expression of the SLC26A4-HA-UTR vector was detected in all ears five weeks post-injection. There were no obvious differences in expression between low and high dose groups. The expression pattern mostly matched the expected endogenous pendrin protein expression pattern, though additional expression of AAV-SLC26A4-HA-UTR was observed in the stria vascularis and outer sulcus cells.

[0242] This study show s that AAV-SLC26A4-HA-UTR is able to express pendrin in DFNB4- relevant cell ty pes in the NHP inner ear.* * *

[0243] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0244] All references (e g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) w as specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0245] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0246] Other embodiments are within the following claims.SEQUENCES

Claims

CLAIMSWhat is claimed is:

1. A polynucleotide comprising from 5' to 3': a) a pendrin protein coding sequence; and b) an engineered 3' untranslated region (3' UTR) of 15 to about 2,000 nucleotides in length, the engineered 3' UTR comprising two or more non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11.

2. The polynucleotide of claim 1, wherein the pendrin protein is a human pendrin protein.

3. The polynucleotide of claim 2, wherein the pendrin protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1.

4. The polynucleotide of claim 3, wherein the pendrin protein comprises the amino acid sequence of SEQ ID NO: 1.

5. The polynucleotide of any one of claims 1-4, wherein the pendrin protein coding sequence comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 2.

6. The polynucleotide of claim 5, wherein the pendrin protein coding sequence comprises the nucleotide sequence of SEQ ID NO: 2.

7. The polynucleotide of any one of claims 1-6, wherein the engineered 3' UTR comprises two. three, four, five, six, seven, eight, or nine non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11.

8. The polynucleotide of any one of claims 1 -7, wherein the engineered 3' UTR comprises, from 5' to 3': a) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 3; b) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 4; c) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5; d) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6;e) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7; f) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 8; g) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 9; h) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10; and i) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11.

9. The polynucleotide of any one of claims 1-8, wherein the engineered 3' UTR comprises, from 5' to 3': a) a nucleotide sequence consisting of SEQ ID NO: 3; b) a nucleotide sequence consisting of SEQ ID NO: 4; c) a nucleotide sequence consisting of SEQ ID NO: 5; d) a nucleotide sequence consisting of SEQ ID NO: 6; e) a nucleotide sequence consisting of SEQ ID NO: 7; f) a nucleotide sequence consisting of SEQ ID NO: 8; g) a nucleotide sequence consisting of SEQ ID NO: 9; h) a nucleotide sequence consisting of SEQ ID NO: 10; and i) a nucleotide sequence consisting of SEQ ID NO: 11.

10. The polynucleotide of any one of claims 1-9, wherein the engineered 3' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13.

11. The polynucleotide of any one of claims 1-10, wherein the engineered 3' UTR comprises the nucleotide sequence of SEQ ID NO: 13.

12. The polynucleotide of any one of claims 1-11. wherein the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 13.

13. The polynucleotide of any one of claims 1-12, further comprising a 5' untranslated region (5' UTR) positioned 5' of the pendrin protein coding sequence.

14. The polynucleotide of claim 13, wherein the 5' UTR is 15 to about 200 nucleotides in length.

15. The polynucleotide of claim 13 or 14, wherein the 5' UTR comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence set forth in SEQ ID NO: 16.

16. The polynucleotide of any one of claims 13-15, wherein the 5' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 17.

17. The polynucleotide of any one of claims 13-16, wherein the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 17.

18. The polynucleotide of any one of claims 13-17, wherein the 5' UTR consists of the nucleotide sequence of SEQ ID NO: 17.

19. The polynucleotide of any one of claims 1 -18, further comprising a promoter operably linked to the pendrin protein coding sequence.

20. The polynucleotide of claim 19, wherein the promoter is a chicken beta-actin (CBA) promoter, a human SLC26A4 promoter, a CAG promoter, a cytomegalovirus (CMV) promoter, a eukaryotic translation elongation factor la (EFla) promoter, a T7 polymerase promoter, or a simian vacuolating virus 40 (SV40) promoter.

21. The polynucleotide of claim 20, wherein the CBA promoter comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 18 or 19.

22. The polynucleotide of claim 20 or 21, wherein the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 18 or 19.

23. The polynucleotide of any one of claims 20-22, wherein the CBA promoter consists of the nucleotide sequence of SEQ ID NO: 18 or 19.

24. The polynucleotide of any one of claims 19-23, further comprising an intron element positioned between the promoter and the pendrin protein coding sequence.

25. The polynucleotide of claim 24, wherein the intron element is an SV-40 intron element or a CBA intron element.

26. The polynucleotide of claim 24 or 25, wherein the intron element comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 27.

27. The polynucleotide of any one of claims 24-26, wherein the intron element comprises the nucleotide sequence of SEQ ID NO: 27.

28. The polynucleotide of any one of claims 24-27, wherein the intron element consists of the nucleotide sequence of SEQ ID NO: 27.

29. The polynucleotide of any one of claims 19-28, further comprising an enhancer positioned 5' of the promoter.

30. The polynucleotide of claim 29, wherein the enhancer comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 28.

31. The polynucleotide of claim 29 or 30, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO: 28.

32. The polynucleotide of any one of claims 29-31, wherein the enhancer consists of the nucleotide sequence of SEQ ID NO: 28.

33. The polynucleotide of any one of claims 1-32, further comprising a polyadenylation sequence positioned 3' of the engineered 3' UTR.

34. The polynucleotide of claim 33, wherein the polyadenylation sequence is a bovine growth hormone polyadenylation sequence.

35. The polynucleotide of claim 33 or 34, wherein the polyadenylation sequence comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 29.

36. The polynucleotide of any one of claims 33-35, wherein the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 29.

37. The polynucleotide of any one of claims 33-36, wherein the polyadenylation sequence consists of the nucleotide sequence of SEQ ID NO: 29.

38. The polynucleotide of any one of claims 1-37, wherein the polynucleotide is capable of expressing pendrin in cells that normally express the SLC26A4 gene.

39. The polynucleotide of any one of claims 1-38. wherein the polynucleotide is capable of expressing pendrin in inner ear cells.

40. The polynucleotide of any one of claims 1-39, wherein the polynucleotide is capable of expressing pendrin in root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.

41. The polynucleotide of any one of claims 1-40, further comprising an inverted terminal repeat (ITR) positioned at the 5' end (5' ITR) and / or an ITR positioned at the 3' end (3' ITR).

42. The polynucleotide of claim 41, wherein the ITR is derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.

43. The polynucleotide of claim 41 or 42, wherein the ITR is derived from AAV2.

44. The polynucleotide of any one of claims 41-43, wherein the polynucleotide comprises: a) a 5' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 32; and / or b) a 3’ ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 33.

45. The polynucleotide of any one of claims 41-44, wherein the polynucleotide comprises: a) a 5' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 32; and b) a 3' ITR comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 33.

46. The polynucleotide of claim 44 or 45, wherein the 5' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 32.

47. The polynucleotide of any one of claims 44-46, wherein the 3' ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 33.

48. The polynucleotide of any one of claims 1-47. wherein the polynucleotide comprises a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 34, 37, 41, and 42.

49. The polynucleotide of any one of claims 1-48, wherein the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs:

34. 37, 41, and 42.

50. The polynucleotide of any one of claims 1-49, wherein the polynucleotide consists of the nucleotide sequence of any one of SEQ ID NOs:

34.

37.

41. and 42.

51. A vector comprising the polynucleotide of any one of claims 1 -50.

52. The vector of claim 51, wherein the vector is a plasmid or a viral vector.

53. The vector of claim 52, wherein the viral vector is an AAV vector.

54. A recombinant adeno-associated virus (rAAV) comprising: a) a capsid protein; and b) the polynucleotide of any one of claims 1-50.

55. The rAAV of claim 54, wherein the rAAV has tropism for a subset of inner ear cells that normally express the SLC26A4 gene.

56. The rAAV of claim 54 or 55, wherein the rAAV has tropism for cells of the cochlea.

57. The rAAV of any one of claims 54-56, wherein the capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV -PHP. B capsid protein, an AAV-S capsid protein, or a variant thereof.

58. The rAAV of any one of claims 54-57, wherein the capsid protein is an AAV-S capsid protein.

59. A cell comprising the polynucleotide of any one of claims 1-50, the vector of any one of claims 51-53, or the rAAV of any one of claims 54-58.

60. A composition comprising the polynucleotide of any one of claims 1-50, the vector of any one of claims 51-53, the rAAV of any one of claims 54-58, or the cell of claim 59, and a pharmaceutically acceptable excipient.

61. A method for specifically expressing pendrin in cells that normally express the SLC26A4 gene in a subject, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-50, the vector of any one of claims 51- 53, the rAAV of any one of claims 54-58, the cell of claim 59, or the composition of claim 60.

62. A method for treating hearing loss in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1- 50, the vector of any one of claims 51-53, the rAAV of any one of claims 54-58, the cell of claim 59, or the composition of claim 60.

63. The method of claim 62, wherein the hearing loss is genetic hearing loss.

64. The method of claim 62 or 63, wherein the subject has or is suspected to have an enlarged vestibular aqueduct (EVA).

65. The method of any one of claims 62-64, wherein the hearing loss is associated with a mutation in the SLC26A4 gene.

66. The method of any one of claims 62-65, wherein the subject has or is suspected to have Pendred syndrome (PDS) and / or autosomal recessive nonsyndromic deafness 4 (DFNB4).

67. A method for treating a pendrin-associated disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-50, the vector of any one of claims 51-53, the rAAV of any one of claims 54-58, the cell of claim 59, or the composition of claim 60.

68. The method of claim 67, wherein the subject has or is suspected to have an enlarged vestibular aqueduct (EVA).

69. The method of claim 67 or 68. wherein the pendnn-associated disease is Pendred syndrome (PDS) or autosomal recessive nonsyndromic deafness 4 (DFNB4).

70. The method of any one of claims 61-69, wherein the subject is a mammal.

71. The method of claim 70, wherein the mammal is a human.

72. The method of claim 70, wherein the mammal is a non-human mammal.

73. The method of claim 72, wherein the non-human mammal is a mouse, a rat, or a non- human primate.

74. The method of any one of claims 61-73, wherein the step of administering results in expression of pendrin protein in inner ear cells.

75. The method of claim 74, wherein the inner ear cells are root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.

76. The method of any one of claims 61-75, wherein the administration is via injection.

77. The method of claim 76, wherein the injection is through the round window membrane of the cochlea, into the scala media of the cochlea, into the scala vestibuli of the cochlea, into a semicircular canal of the inner ear. or into the saccule or the utricle of the inner ear.

78. A polynucleotide comprising an engineered 3' untranslated region (3' UTR) of 15 to about 2,000 nucleotides in length, the engineered 3' UTR comprising two or more nonidentical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11.

79. The polynucleotide of claim 78, wherein the engineered 3' UTR comprises three, four, five, six, seven, eight, or nine non-identical nucleotide sequences that are each at least 80% identical to any one of SEQ ID NOs: 3-11.

80. The polynucleotide of claim 78 or 79, wherein the engineered 3' UTR comprises, from 5' to 3': a) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 3; b) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 4; c) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5; d) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6; e) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7; f) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 8; g) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 9; h) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10; and i) a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11.

81. The polynucleotide of any one of claims 78-80, wherein the engineered 3' UTR comprises, from 5' to 3': a) a nucleotide sequence consisting of SEQ ID NO: 3; b) a nucleotide sequence consisting of SEQ ID NO: 4; c) a nucleotide sequence consisting of SEQ ID NO: 5; d) a nucleotide sequence consisting of SEQ ID NO: 6; e) a nucleotide sequence consisting of SEQ ID NO: 7;I) a nucleotide sequence consisting of SEQ ID NO: 8; g) a nucleotide sequence consisting of SEQ ID NO: 9; h) a nucleotide sequence consisting of SEQ ID NO: 10; and i) a nucleotide sequence consisting of SEQ ID NO: 11.

82. The polynucleotide of any one of claims 78-81, wherein the engineered 3' UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13.

83. The polynucleotide of any one of claims 78-82, wherein the engineered 3' UTR comprises the nucleotide sequence of SEQ ID NO: 13.

84. The polynucleotide of any one of claims 78-83, wherein the engineered 3' UTR consists of the nucleotide sequence of SEQ ID NO: 13.

85. The polynucleotide of any one of claims 78-84, further comprising a transgene positioned 5' of the engineered 3' UTR.

86. The polynucleotide of claim 85, further comprising a 5' untranslated region (5' UTR) positioned 5' of the transgene.

87. The polynucleotide of claim 86, wherein the 5' UTR is 15 to about 200 nucleotides in length.

88. The polynucleotide of claim 86 or 87, wherein the 5' UTR comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence set forth in SEQ ID NO: 16.

89. The polynucleotide of any one of claims 86-88, wherein the 5’ UTR comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 17.

90. The polynucleotide of any one of claims 86-89, wherein the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 17.

91. The polynucleotide of any one of claims 86-90, wherein the 5' UTR consists of the nucleotide sequence of SEQ ID NO: 17.

92. The polynucleotide of any one of claims 85-91, further comprising a promoter operably linked to the transgene.

93. The polynucleotide of any one of claims 85-92, wherein the polynucleotide is capable of expressing the transgene in inner ear cells.

94. The polynucleotide of any one of claims 85-93, wherein the polynucleotide is capable of expressing the transgene in root cells, spindle cells, epithelial cells of the spiral prominence, outer sulcus cells, Hensen’s cells, Claudius cells, spiral ganglion neurons, cells of the endolymphatic sac, and / or vestibular cells.

95. The polynucleotide of any one of claims 78-94, further comprising an inverted terminal repeat (ITR) positioned at the 5' end (5’ ITR) and / or an ITR positioned at the 3' end (3' ITR).

96. A vector comprising the polynucleotide of any one of claims 78-95.

97. The vector of claim 96, wherein the vector is a plasmid or a viral vector.

98. The vector of claim 97, wherein the viral vector is an AAV vector.

99. A recombinant adeno-associated virus (rAAV) comprising: a) a capsid protein; and b) the polynucleotide of any one of claims 78-95.

100. The rAAV of claim 99, wherein the rAAV has tropism for inner ear cells.

101. The rAAV of claim 99 or 100, wherein the rAAV has tropism for cells of the cochlea.

102. The rAAV of any one of claims 99-101, wherein the capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV -PHP. B capsid protein, an AAV-S capsid protein, or a variant thereof.

103. The rAAV of any one of claims 99-102, wherein the capsid protein is an AAV-S capsid protein.

104. A cell comprising the polynucleotide of any one of claims 78-95, the vector of any one of claims 96-98, or the rAAV of any one of claims 99-103.

105. A composition comprising the polynucleotide of any one of claims 78-95, the vector of any one of claims 96-98, the rAAV of any one of claims 99-103, or the cell of claim 104, and a pharmaceutically acceptable excipient.

106. A method for specifically expressing a transgene in inner ear cells in a subject, the method comprising administering to the subject an effective amount of the polynucleotide ofany one of claims 85-95, the vector of any one of claims 96-98, the rAAV of any one of claims 99-103, the cell of claim 104. or the composition of claim 105.

Images