Cell-type-specific control of gene expression in auditory hair cells

Abstract

Provided herein are chimeric minigenes, where the alternative splicing of the minigene ensures that an encoded transgene is expressed only in auditory hair cells. The encoded transgene may encode an inhibitory RNA, a CRISPR-Cas9 protein, a transactivator, or a therapeutic protein. Also provided are AAVs comprising the chimeric minigene and having modified capsid proteins that target the cochlea.

Description

[0001]PATENT CHOP.P0075WO CELL-TYPE-SPECIFIC CONTROL OF GENE EXPRESSION IN AUDITORY HAIR CELLS REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of United States provisional application number 63 / 549,784, filed February 5, 2024, the entire contents of which are incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on February 5, 2025, is named CHOPP0075WO.xml and is 36,149 bytes in size. BACKGROUND 1. Field The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns compositions and methods for using alternative splicing regulation to modulate expression of a therapeutic gene in auditory hair cells. 2. Description of Related Art While viral and nonviral approaches for gene therapies have made tremendous advancements over the last twenty years, the major focus has been on the cargo delivery system; e.g., viral capsid evolution and engineering for adeno-associated viruses (AAVs), expanding the landscape of cell-targeting envelopes for lentiviruses, and refining lipid nanoparticles for improved uptake. However, the cargo itself, and more importantly the elements controlling the expression from that cargo, have been largely untouched aside from using engineered promoters or 3’ regulatory elements to restrict expression to certain cell types (Brown et al., 2006; Domenger & Grimm, 2019). As such, compositions and methods for modulating expression of therapeutic genes in cargo delivery systems are needed. SUMMARY Provided herein are compositions and methods for restricting expression of a transgene to auditory hair cells using an alternative splicing switch. These compositions and methods do not require any bacterial or other external elements for regulation. Provided herein are compositions for use in the treatment of genetic hearing loss or for inducing auditory hair cell regeneration, the compositions comprising a nucleic acid molecule comprising a first expression cassette comprising, from 5’ to 3’, (a) a minigene having an alternatively spliced exon that is only included in a cochlear cell type and (b) an encoded therapeutic transgene, such that the encoded therapeutic transgene is only expressed in the cochlear cell upon inclusion of the alternatively spliced exon. In some aspects, the minigene comprises, from 5’ to 3’, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3, wherein Exon 2 is the alternatively spliced exon that is only included in a cochlear cell type, and wherein Exon 2 comprises translation initiation regulatory sequences. In some aspects, the nucleic acid molecule is comprised in a recombinant adeno-associated virus (rAAV). Provided herein are nucleic acid molecules comprising a first expression cassette comprising, from 5’ to 3’, (a) a minigene having an alternatively spliced exon and (b) an encoded transgene; wherein the minigene comprises, from 5’ to 3’, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3, wherein Exon 2 is the alternatively spliced exon, wherein Exon 2 is derived from Exon 18C of the human Esp8 gene, and wherein Exon 2 comprises translation initiation regulatory sequences. Exon 2 may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 2. The encoded transgene is in frame with the translation initiation regulatory sequence in Exon 2. Exon 1 may be derived from Exon 18 of the human Esp8 gene. Exon 1 may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1. Exon 3 may be derived from Exon 19 of the human Esp8 gene. Exon 3 may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 3. Intron 1 may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence 2 4898-4878-0311, v.1 of SEQ ID NO: 4. Intron 2 may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 5. The minigene may comprise or consist of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 6. The expression of the encoded transgene may not require the co-expression of any exogenous regulatory protein. The encoded transgene may encode a therapeutic protein, a Cas9 protein, or a transactivator protein. The therapeutic protein may be a protein whose deficiency is associated with hearing loss. The encoded transgene may not be a reporter. The minigene and the encoded gene may be separated by a cleavable peptide. The first expression cassette may be operably linked to a first promoter. The first promoter may be a constitutive promoter. The first promoter may be a Rous sarcoma virus (RSV) promoter, the phosphoglycerate kinase (PGK) promoter, a JeT promoter, a CBA promoter, a synapsin promoter, or the minimal cytomegalovirus (mCMV) promoter. The therapeutic transgene may be operably linked to a cochlea-specific promoter. The cochlea-specific promoter may be an auditory hair cell-specific or -selective promoter. The nucleic acid molecules may further comprise a second expression cassette. The second expression cassette may comprise a nucleic acid sequence encoding a guide RNA operably linked to a second promoter. The second expression cassette may comprise a nucleic acid sequence encoding a therapeutic protein, an inhibitory RNA, or a Cas9 protein, wherein the nucleic acid sequence is operably linked to a second promoter, wherein the second promoter is activated by the transactivator encoded by the first expression cassette. The inhibitory RNA may be a siRNA, shRNA, or miRNA. The inhibitory RNA may inhibit or decrease expression of an aberrant or abnormal protein associated with hearing loss. Provided herein are cells comprising the nucleic acid molecule of any one of the present embodiments. Provided herein are recombinant adeno-associated viruses (rAAV) comprising an AAV capsid protein and nucleic acid molecule provided herein. The rAAV capsid protein may be a modified rAAV capsid protein described herein. 3 4898-4878-0311, v.1 Provided herein are pharmaceutical compositions comprising the rAAV provided herein and a pharmaceutically acceptable carrier Provided herein are methods of administering the encoded transgene to a patient in need thereof, the method comprising administering the nucleic acid, the rAAV, or the pharmaceutical composition provided herein to the patient. Provided herein are method of treating genetic hearing loss or for inducing auditory hair cell regeneration, the methods comprising administering to a patient in need thereof a nucleic acid molecule comprising a first expression cassette comprising, from 5’ to 3’, (a) a minigene having an alternatively spliced exon that is only included in a cochlear cell type and (b) an encoded therapeutic transgene, such that the encoded therapeutic transgene is only expressed in the cochlear cell. In some aspects, the minigene comprises, from 5’ to 3’, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3, wherein Exon 2 is the alternatively spliced exon that is only included in a cochlear cell type, and wherein Exon 2 comprises translation initiation regulatory sequences. In some aspects, the nucleic acid molecule is comprised in a recombinant adeno-associated virus (rAAV). In some aspects, the rAAV vector comprises an AAV particle comprising AAV capsid proteins, and wherein the first and / or second expression cassette is inserted between a pair of AAV inverted terminal repeats (ITRs). In some aspects, the rAAV is a self- complementary AAV (scAAV) vector. In some aspects, the rAAV is a single-stranded AAV (ssAAV). In some aspects, the AAV capsid proteins are derived from or selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8 VP1, VP2 and / or VP3 capsid proteins, or a capsid protein having 70% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV- Rh10, or AAV-2i8 VP1, VP2 and / or VP3 capsid proteins. In some aspects, the pair of AAV ITRs is derived from, comprises or consists of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or AAV- 2i8 ITR, or an ITR having 70% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10, or AAV- 2i8 ITR sequence. 4 4898-4878-0311, v.1 In some aspects, a plurality of the viral vectors are administered. In some aspects, the viral vectors are administered at a dose of about 1×106to about 1×1018vector genomes per kilogram of the patient (vg / kg). In some aspects, the viral vectors are administered at a dose from about 1x107-1x1017, about 1x108-1x1016, about 1x109-1x1015, about 1x1010-1x1014, about 1x1010-1x1013, about 1x1010-1x1013, about 1x1010-1x1011, about 1x1011-1x1012, about 1x1012-x1013, or about 1x1013-1X1014vg / kg of the patient. In some aspects, the viral vectors are administered at a dose of about 0.5-4 ml of 1x106-1x1016vg / ml. In some aspects, the methods further comprise administering a plurality of empty viral capsids. In some aspects, the empty viral capsids are formulated with the viral particles administered to the patient. In some aspects, the empty viral capsids are administered or formulated with 1.0 to 100-fold excess of viral vector particles or empty viral capsids. In some aspects, the empty viral capsids are administered or formulated with 1.0 to 100-fold excess of viral vector particles to empty viral capsids. In some aspects, the empty viral capsids are administered or formulated with about 1.0 to 100-fold excess of empty viral capsids to viral vector particles. The administration may be to the cerebrospinal fluid of the patient. The administration may comprise intracerebroventricular administration. The method may deliver the therapeutic transgene to the spiral organ of Corti. The method may deliver the therapeutic transgene to a cell of the inner ear, such as, for example, an auditory hair cell. The method may treat or prevent hearing loss. The patient may have a hearing disorder, and the molecular therapeutic may be delivered in a therapeutically effective amount. The method may reverse or prevent hearing loss. The hearing loss may be partial hearing loss or complete deafness. The method may treat or prevent hearing loss in a patient. The method may treat hereditary hearing loss in the patient. In some aspects, the patient is a human. In some aspects, the methods further comprise administering one or more immunosuppressive agents. In some aspects, the immunosuppressive agent is administered prior to or contemporaneously with administration of the expression cassettes. In some aspects, the immunosuppressive agent is an anti-inflammatory agent. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the 5 4898-4878-0311, v.1 detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS. 1A-1C. Eps8-derived splicing cassette restricts AAV payload expression to auditory hair cells. (FIG. 1A) IGV (Integrated Genomics Viewer) genome browser screen showing two novel exons (18B and 18C) in the murine and human Eps8 genes. Both the indicated exons (18B and 18C) have high conservation between species with a 100% sequence conservation between mouse and human genomes. In Intropolis, a database of splicing events across the human genome accumulated from RNA-seq datasets across the sequence read archive, the splice junctions giving rise to exon 18C were detected in 18 and 7 datasets at a total read count level of 21 and 8, respectively. In contrast, the canonical Eps8 exon 18-19 junction was observed in 10,613 datasets at a total read count level of 322,438. (FIG. 1B) A schematic description of two fluorescence reporter expression constructs. Construct “18C” is driven by the ubiquitous CAG promoter and contains an exon and splice donor and acceptor sites derived from Eps8 exon 18C along with a Kozak sequence and ATG start codon. The control construct is also driven by the CAG promoter and does not contain a splice junction. These constructs were packaged into “AAV-EAR1” an AAV with extensive tropism for inner hair cells in the murine cochlear basal turn. AAVs were then pooled and infused intracerebroventricularly (ICV) via bilateral injections into the ventricle of three mice (E52, J33, and J35) to achieve transduction of both brain and cochlear cells. (FIG. 1C) Fluorescence images showing signal from Hoechst labeled nuclei, AAV-EAR1.CAG, and AAV-EAR1.EPS8-18c. Regions of the cochlear basal turn containing continuous stretches of auditory inner hair cells are shown from one ear of each mouse in the first three rows. Transduction was bilateral and these images are representative of transduction achieved in the opposite ear. Rows 4-9 show brain images from regions including temporal cortex (tmpCx), motor cortex (mCx), and hippocampus (CA1, CA2, and CA3). Brain images were collected 6 4898-4878-0311, v.1 from 2 of 3 animals. The brain of the third animal is being reserved for FISH staining. White “<” characters indicate mRuby3 positive cells detected in brain. The only observed mRuby3 expressing cells in the brain were observed near to the needle track in one animal. DETAILED DESCRIPTION The recent clinical success of AAV-based gene therapy has demonstrated the feasibility and value of gene therapies for hearing and deafness. Current approaches to cochlear gene therapy use AAV intracochlear infusion via an inner ear surgical approach. This method necessitates general anesthesia, perforation(s) of the sealed inner ear space, and patients of sufficient age to facilitate the approach. However, the complexity of the auditory system and extreme diversity of genetic causes of hearing loss remain barriers to broader targeting of most hearing loss genes. Delivery continues to pose challenges at every level: the therapeutic window of opportunity, the vectors to target various hearing-relevant cochlear cell types, and payload design that limit expression to therapeutically relevant cells. The cerebrospinal fluid (CSF)-mediated route of gene therapy administration to the inner ear and round window membrane combined with canalostomy (RWM) infusions of WT and modified AAVs into NHPs have been developed to identify new hearing-relevant AAV capsids that target primate cochlear cells. The inventors have leveraged this platform for the development of a pipeline for splicing-based regulatory element discovery with validation in murine auditory inner hair cells (IHCs). CSF-mediated delivery of the top modified AAV variants yielded capsids capable of nearly complete IHC transduction across all cochlear turns, robust transduction of spiral ganglion neurons, and transduction of non-sensory supporting cells in the organ of Corti of the NHP cochlea. Additionally, using available single-cell RNA-Seq data, a hair- cell-specific splice event with 100% sequence conservation between mouse and human was tested for its ability to confer hair cell-specific payload expression. After ICV administration to mouse CSF, there was auditory hair cell-specific gene expression, and no expression throughout the non-cochlear central nervous system. As such, provided herein are compositions and methods for gene delivery to the inner ear using regulatory elements to limit gene expression to a cochlear cell-type of interest, providing for gene therapy for hearing loss. 7 4898-4878-0311, v.1 I. Alternative splicing-regulated transgene expression Disclosed herein are chimeric minigenes, where the alternative splicing of the minigene determines whether the downstream encoded transgene is expressed in a cell type- specific manner. The encoded transgene may be an inhibitory RNA, a CRISPR-Cas9 protein, a therapeutic protein, or a transactivator. The minigenes comprise three exons, Exons 1-3, where Exon 2 is skipped in cells other than auditory hair cells. When Exon 2 is skipped, the downstream encoded gene is not produced because the translation initiation regulatory sequences are located in Exon 2. As such, translation of the encoded protein is not initiated. In order for expression of the encoded gene to occur, the inclusion of the skipped exon must be induced. Such can occur only in auditory hair cells. For example, the minigene may comprise Exons 18, 18C, and 19 of the Esp8 gene, in which case Exon 18C is skipped in cells other than auditory hair cells. As such, the downstream encoded gene will be expressed only in auditory hair cells. Exon 1 of the construct may correspond to Exon 18 of the Esp8 gene and have the sequence: 5’-TCCAGAATCTGGATTGGGGCGTGCTGATCCACCTTATACTCATACTATACAG-3’ (SEQ ID NO: 1) Exon 2 of the construct may correspond to Exon 18B or 18C of the Esp8 gene. Exon 18C of the Esp8 gene may correspond to genomic position chr6:137,493,548- 137,493,571 of the mouse genome (mm10) or genomic position chr12:15,787,673- 15,787,696 of the human genome (hg19). Exon 18B of the Esp8 gene may correspond to genomic position chr6:137,498,345-137,498,380 or genomic position chr12:15,792,360- 15,792,395 of the human genome (hg19). Exon 2 of the construct may comprise a Kozak sequence and ATG start codon. Exon 2 may have the sequence: 5’-CAATTACTTGGCGAGATGTCAGAG-3’ (SEQ ID NO: 2) Exon 3 of the construct may correspond to Exon 19 of the Esp8 gene and have the sequence: 5’-AAACAAAGGTTGGAGTTTGGCCCAAGACCAGCTGATACTC-3’ (SEQ ID NO: 3) Intron 1 of the construct, which is positioned between Exon 1 and Exon 2, may have the sequence: 8 4898-4878-0311, v.1 5’- gtgagcatgatttcttagtaaaatgtagtatgtacacatttacgaagttatcactcttaagt aggatttaaacacctttgtgtattgatcattttgagatgtagttggttatatattttttcta gagtaattggtgggggagtggcaatggtgagagacacagggaagaattacacatatgttttc tgatttacttctagaaattgttcaagatgtttagcagtaggtacctgattctctgaatgctt ttttactgttgaagtactttacacggtttctactatagtagaatctatatgattagttttcc atggctagatagtgatgggaattaattatagatttacaggctccttcactggttgattgtgg gcatgtaattttactgtcaggctaatttactctttctgatatttgatgtgaattataattga tattattttaattccatttcctgtattaattttctcatttgatgtgatttgctccattttcc taatag-3’ (SEQ ID NO: 4) Intron 2 of the construct, which is positioned between Exon 2 and Exon 3, may have the sequence: 5’- gtaacccacattcgttccctcatggcacttctttttaatataattttttggattcagtctct caacttaacaccctccaaatgttccagctgtgcttagttttcactgcagagttcccaaataa ccaaatttagaggaaaaatgtgtaatttagagtaggttaaatatttaagaacagttcaattt taatggctttgttgcgcttacttgcataatttcccctctgaagcagcatcttgtatgaatca ggataatactactatttgctattgacgtagccttgtgactccttaactaatgttttgacttt gaacaaattatgtagaatgtttaaaatatgatttttaatggtcattataccatataggatta cagataaatgagtaatgggtttgcaagtaagtttaaaaatagccaacaatgtcctatcaaag tataatttgttttcctagcctaggtttatattttttaaattttaagttaattatttgtcttt ttatag-3’ (SEQ ID NO: 5) The ex18C cassette sequence may be as follows: 5’- TCCAGAATCTGGATTGGGGCGTGCTGATCCACCTTATACTCATACTATACAGgtgagcatga tttcttagtaaaatgtagtatgtacacatttacgaagttatcactcttaagtaggatttaaa cacctttgtgtattgatcattttgagatgtagttggttatatattttttctagagtaattgg tgggggagtggcaatggtgagagacacagggaagaattacacatatgttttctgatttactt ctagaaattgttcaagatgtttagcagtaggtacctgattctctgaatgcttttttactgtt gaagtactttacacggtttctactatagtagaatctatatgattagttttccatggctagat agtgatgggaattaattatagatttacaggctccttcactggttgattgtgggcatgtaatt 9 4898-4878-0311, v.1 ttactgtcaggctaatttactctttctgatatttgatgtgaattataattgatattatttta attccatttcctgtattaattttctcatttgatgtgatttgctccattttcctaatagCAAT TACTTGGCGAGATGTCAGAGgtaacccacattcgttccctcatggcacttctttttaatata attttttggattcagtctctcaacttaacaccctccaaatgttccagctgtgcttagttttc actgcagagttcccaaataaccaaatttagaggaaaaatgtgtaatttagagtaggttaaat atttaagaacagttcaattttaatggctttgttgcgcttacttgcataatttcccctctgaa gcagcatcttgtatgaatcaggataatactactatttgctattgacgtagccttgtgactcc ttaactaatgttttgactttgaacaaattatgtagaatgtttaaaatatgatttttaatggt cattataccatataggattacagataaatgagtaatgggtttgcaagtaagtttaaaaatag ccaacaatgtcctatcaaagtataatttgttttcctagcctaggtttatattttttaaattt taagttaattatttgtctttttatagAAACAAAGGTTGGAGTTTGGCCCAAGACCAGCTGAT ACTC-3’ (SEQ ID NO: 6) The coding sequence for the encoded transgene is placed 3' of the Exon 3 sequence of SEQ ID NO: 3 or of the cassette sequence of SEQ ID NO: 6. The expression of the chimeric minigene may be regulated by various types of promoters, depending on the desired expression pattern. For example, the promoter may be a universally constitutive promoter, such as a promoter for a housekeeping gene (e.g., ACTB) or the ubiquitous CAG promoter. As another example, the promoter may be a cell-type specific promoter for auditory hair cells. As yet another example, the promoter may be an inducible promoter. The chimeric minigene may have a cleavable peptide located between the minigene and the encoded transgene. In some cases, the cleavable peptide may be a self- cleavable peptide, such as, for example, a 2A peptide. The 2A peptide may be a T2A peptide, a P2A peptide, an E2A peptide, or a F2A peptide. The presence of this peptide provides for separation of the minigene-encoded peptide from the encoded protein following translation. In some cases, the cleavable peptide may be a cleavage site for a widely expressed, endogenous endoprotease, such as, for example, furin, prohormone convertase 7 (PC7), paired basic amino-acid cleaving enzyme 4 (PACE4), or subtilisin kexin isozyme 2 (SKI-1). In some cases, the cleavable peptide may be a cleavage site for a tissue-specific or cell- specific endoprotease (such as, e.g., prohormone convertase 2 (PC2; primarily expressed in endocrine tissue and brain), prohormone convertase 1 / 3 (PC1 / 3; primarily expressed in endocrine tissue and brain), prohormone convertase 4 (PC4; primarily expressed in the testis 10 4898-4878-0311, v.1 and ovary), and proprotein convertase subtilisin kexin 9 (PSCK9; primarily expressed in the lung and liver)). II. Exemplary encoded transgenes for treatment of hearing loss In some embodiments, viral gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding therapeutic proteins to cells in culture or in a host organism. A. CRISPR Systems Gene editing is a technology that allows for the modification of target genes within living cells. Recently, harnessing the bacterial immune system of CRISPR to perform on demand gene editing revolutionized the way scientists approach genomic editing. The Cas9 protein of the CRISPR system, which is an RNA guided DNA endonuclease, can be engineered to target new sites with relative ease by altering its guide RNA sequence. This discovery has made sequence specific gene editing functionally effective. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus. The CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non- coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting 11 4898-4878-0311, v.1 sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor (e.g., KRAB) or activator, to affect gene expression. Alternatively, a CRISPR system with a catalytically inactivate Cas9 further comprises a transcriptional repressor or activator fused to a ribosomal binding protein. In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence.” In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination. Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more 12 4898-4878-0311, v.1 nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. The tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. The Cas enzyme may be a target gene under the control of a regulated alternative splicing event, as disclosed herein, either as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA may be under the control of a constitutive promoter. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. A vector may comprise a regulatory element operably linked to an enzyme- coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the 13 4898-4878-0311, v.1 amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR. In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some 14 4898-4878-0311, v.1 embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference. 15 4898-4878-0311, v.1 B. Therapeutic Proteins Some embodiments concern expression of recombinant proteins and polypeptides. Such proteins may be otoprotective proteins, such as an anti-apoptotic protein, an anti-oxidant enzyme (e.g., those belonging to the superoxide dismutase (SOD) family), a neurotrophic / neuroprotective factor, an anti-inflammatory protein, or a protein that promoters hair cells regeneration in the vestibular system. The therapeutic protein may be Birc1a (NAIP), Birc2 (c-IAP1 / HIAP-2), Birc3 (cIAP-2 / HIAP-1), Birc4 (XIAP), Birc5 (survivin), Birc6 (apollon), Birc7 (livin), Birc8 (TsIAP); members of the Bcl-2 family: Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bcl-2L10, BFL-1; endogenous inhibitors of the c-Jun N-terminus kinase (JNK) known as Jun-interacting protein (JIP), JIP-1, JIP-2, JIP-3, JIP-4; SOD1, SOD2; catalase; peroxiredoxin-1, peroxiredoxin-2, glutathione preoxidase 1 (Gpx1), Gpx2, Gpx3, or Gpx4; NGF, BDNF, CNTF, GDNF, Growth / differentiation factor-15 (GDF-15), erythropoietin or vascular endothelial growth factor (VEGF); interleukin-10 (IL-10); glutathione S-transferase, Annexin-1 (ANXA1), inhibitor of NF-κB (IκB); USH1, USH1G (a.k.a. SANS), CIB2 (calcium and integrin binding protein 2), ATOH-1, ACTG1, ATP2B2, CDH23 (cadherin 23), CLDN14, CLRN1, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB2, GJB3, GJB6, KCNQ4, LHFPL5, MT-RNR1, MT-TS1, MYO1A, MYO6, MYO7A (myosin 7a), MYO15A, OTOF, PCDH15 (protocadherin 15), PDZD7, POU3F4, SLC26A4, STRC, TECTA, TMC1, TMC2, TMIE, TMPRSS3, TRIOBP, USH1C, VLGR1, WFS1, ACTG1, ADCY1, ATOFfl, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD 164, CDC 14 A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRNl, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A 1, COL9A2, COL11 A1, COL11 A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1 , GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS 1, MYH14, ΜΥΉ9, MY015A, MYOIA, MY03A, MY06, MY07A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS 1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, S MAC / DIABLO, SNAI2, SOX 10, STRC, SYNE4, TBC 1 D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM 132E, TMPRSS3, 16 4898-4878-0311, v.1 TRPN, TRIOBP, TSPEAR, USH1 C, USH1 G, USH2A, USH2D, VLGRl, WFS1, WHRN, or XIAP. Further exemplary therapeutic proteins and the associated conditions are listed in Table A. Table A. NCBI RefSeq ID Disease or condition Gene GENE Protein C / V N N Additional conditions and the associated genetic defects have been described in the art; see, e.g., Kemperman et al., J R Soc Med. 2002 Apr; 95(4): 171-177; Gazquez and Lopez-Escamez, Curr Genomics. 2011 Sep; 12(6): 443-450; Duan et al., Gene Therapy (2004) 11:S51-S56. When the present application refers to the function or activity of “modified protein” or a “modified polypeptide,” one of ordinary skill in the art would understand that this includes, for example, a protein or polypeptide that possesses an additional advantage over the unmodified protein or polypeptide. It is specifically contemplated that embodiments 17 4898-4878-0311, v.1 concerning a “modified protein” may be implemented with respect to a “modified polypeptide,” and vice versa. Recombinant proteins may possess deletions and / or substitutions of amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments, these proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example. A “modified deleted protein” lacks one or more residues of the native protein, but may possess the specificity and / or activity of the native protein. A “modified deleted protein” may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one that has an amino acid residue deleted from at least one antigenic region, i.e. a region of the protein determined to be antigenic in a particular organism, such as the organism to which the modified protein is being administered. Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. In addition to a deletion or substitution, a modified protein may possess an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, called fusion proteins, are discussed below. The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 18 4898-4878-0311, v.1 99% of amino acids that are identical or functionally equivalent to the amino acids of a control polypeptide are included, provided the biological activity of the protein is maintained. A recombinant protein may be biologically functionally equivalent to its native counterpart in certain aspects. It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. As used herein, a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full-length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and / or a peptide of from about 3 to about 100 amino acids. For convenience, the terms “protein,” “polypeptide,” and “peptide are used interchangeably herein. As used herein, an “amino acid residue” refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are sequential, without any non-amino acids interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties. Accordingly, the term “protein or peptide” encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid. Certain embodiments of the present invention concern fusion proteins. These molecules may have a therapeutic protein linked at the N- or C-terminus to a heterologous domain. For example, fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion 19 4898-4878-0311, v.1 includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein. Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the polypeptides connected in tandem. The linker would be of sufficient length to allow proper folding of the resulting fusion protein. Expression of a transgene may be directed by the transgene’s natural promoter (i.e., the promoter found naturally with the transgenic coding sequence) or expression of a transgene may be directed by a heterologous promoter (e.g., CMV promoter, Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter). For example, any of the transgenes described herein can be used with its natural promoter. Alternatively, any of the transgenes described herein can be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally direct expression of that sequence (i.e., is not found with that sequence in nature). Representative heterologous promoters that can be used to direct expression of any of the transgenes indicated herein include, for example, a CMV promoter, a CBA promoter, a CASI promoter, a P promoter, and a EF-1 promoter, an alpha9 nicotinic receptor promoter, a prestin promoter, a Gfil promoter, and a Vglut3 promoter. In addition, a promoter that naturally directs expression of one of the above-referenced transgenes (e.g., a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter or an Atohl promoter) can be used as a heterologous promoter to direct expression of a transgene. In other embodiments, the promoter is an Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter. 20 4898-4878-0311, v.1 III. Viral Vectors Gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNAs, therapeutic proteins, or components of a CRISPR system to cells in culture, or in a host organism. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include exosomes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent- enhanced uptake of DNA. Lipofection is described in (e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91117424; WO 91116024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). In some embodiments, delivery is via the use of RNA or DNA viral based systems for the delivery of nucleic acids. Viral vectors may be administered directly to patients (in vivo) or they can be used to treat cells in vitro or ex vivo, and then administered to patients. Viral-based systems in some embodiments include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control element (e.g., a promoter, enhancer), intron, ITR(s), and polyadenylation signal. 21 4898-4878-0311, v.1 A viral vector is derived from or based upon one or more nucleic acid elements that comprise a viral genome. Exemplary viral vectors include adeno-associated virus (AAV) vectors, retroviral vectors, and lentiviral vectors. The term “recombinant,” as a modifier of vector, such as recombinant viral, e.g., lenti- or parvo-virus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted within the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into a vector, with or without 5ʹ, 3ʹ and / or intron regions that the gene is normally associated within the viral genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission. A recombinant viral “vector” is derived from the wild type genome of a virus, such as AAV, retrovirus, or lentivirus, by using molecular methods to remove the wild type genome from the virus, and replacing with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A “recombinant” viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome, since all or a part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.” Adeno-associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV is distinct from other members of this family by its dependence upon a helper virus for replication. 22 4898-4878-0311, v.1 AAV genomes can exist in an extrachromosomal state without integrating into host cellular genomes; possess a broad host range; transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes. AAV viral particles are heat stable; resistant to solvents, detergents, changes in pH, and temperature; and can be column purified and / or concentrated on CsCl gradients or by other means. The AAV genome comprises a single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The approximately 5 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. The ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as the origin of viral DNA replication. An AAV “genome” refers to a recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. An AAV particle often comprises an AAV genome packaged with AAV capsid proteins. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the AAV vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsulated into viral particles. Thus, an AAV vector “genome” refers to nucleic acid that is packaged or encapsulated by AAV capsid proteins. The AAV virion (particle) is a non-enveloped, icosahedral particle approximately 25 nm in diameter. The AAV particle comprises an icosahedral symmetry comprised of three related capsid proteins, VP1, VP2 and VP3, which interact together to form the capsid. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10 respectively, but may be in varied ratios, and are all derived from the right-hand ORF. The VP1, VP2 and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1 which is translated from an alternatively spliced message results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any single-stranded progeny DNA or infectious 23 4898-4878-0311, v.1 particles. In certain embodiments, the genome of an AAV particle encodes one, two or all VP1, VP2 and VP3 polypeptides. The genome of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF. The left ORF often encodes the non-structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68 / 78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities. Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. In certain embodiments the genome of an AAV (e.g., an rAAV) encodes some or all of the Rep proteins. In certain embodiments the genome of an AAV (e.g., an rAAV) does not encode the Rep proteins. In certain embodiments one or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a polypeptide. The ends of the AAV genome comprise short inverted terminal repeats (ITR) which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Accordingly, the genome of an AAV comprises one or more (e.g., a pair of) ITR sequences that flank a single stranded viral DNA genome. The ITR sequences often have a length of about 145 bases each. Within the ITR region, two elements have been described which are believed to be central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration. The term “recombinant,” as a modifier of vector, such as recombinant viral, e.g., lenti- or parvo-virus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector 24 4898-4878-0311, v.1 would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted within the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into a vector, with or without 5ʹ, 3ʹ and / or intron regions that the gene is normally associated within the viral genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission. A recombinant viral “vector” is derived from the wild type genome of a virus by using molecular methods to remove part of the wild type genome from the virus, and replacing with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A “recombinant” viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome, since part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.” In certain embodiments, an AAV (e.g., a rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs that flank (i.e., are at each 5ʹ and 3ʹ end) of a nucleic acid sequence that at least encodes a polypeptide having function or activity. An AAV vector (e.g., rAAV vector) can be packaged and is referred to herein as an “AAV particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle can also be referred to as a “rAAV particle.” In certain embodiments, an AAV particle is a rAAV particle. A rAAV particle often comprises a rAAV vector, or a portion thereof. A rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles). rAAV particles typically comprise proteins that encapsulate or package the rAAV 25 4898-4878-0311, v.1 vector genome (e.g., capsid proteins). It is noted that reference to a rAAV vector can also be used to reference a rAAV particle. Any suitable AAV particle (e.g., rAAV particle) can be used for a method or use herein. A rAAV particle, and / or genome comprised therein, can be derived from any suitable serotype or strain of AAV. A rAAV particle, and / or genome comprised therein, can be derived from two or more serotypes or strains of AAV. Accordingly, a rAAV can comprise proteins and / or nucleic acids, or portions thereof, of any serotype or strain of AAV, wherein the AAV particle is suitable for infection and / or transduction of a mammalian cell. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV- 2i8. In certain embodiments a plurality of rAAV particles comprises particles of, or derived from, the same strain or serotype (or subgroup or variant). In certain embodiments a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains). As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype. In certain embodiments, a rAAV vector based upon a first serotype genome corresponds to the serotype of one or more of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that comprises the AAV vector genome corresponds to the serotype of a capsid that comprises the rAAV particle. In certain embodiments, a rAAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from the serotype of one or more of the AAV capsid proteins that package the vector. For example, a rAAV vector genome can comprise AAV2 26 4898-4878-0311, v.1 derived nucleic acids (e.g., ITRs), whereas at least one or more of the three capsid proteins are derived from a different serotype, e.g., an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype or variant thereof. In certain embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 particle. In particular embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype. In certain embodiments, a method herein comprises use, administration or delivery of a rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74 or rAAV-2i8 particle. In certain embodiments, a method herein comprises use, administration or delivery of a rAAV2 particle. In certain embodiments a rAAV2 particle comprises an AAV2 capsid. In certain embodiments a rAAV2 particle comprises one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV2 particle. In certain embodiments a rAAV2 particle comprises VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV2 particle. In certain embodiments, a rAAV2 particle is a variant of a native or wild-type AAV2 particle. In some aspects, one or more capsid 27 4898-4878-0311, v.1 proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to capsid protein(s) of a native or wild-type AAV2 particle. In certain embodiments a rAAV9 particle comprises an AAV9 capsid. In certain embodiments a rAAV9 particle comprises one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV9 particle. In certain embodiments a rAAV9 particle comprises VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV9 particle. In certain embodiments, a rAAV9 particle is a variant of a native or wild-type AAV9 particle. In some aspects, one or more capsid proteins of an AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to capsid protein(s) of a native or wild-type AAV9 particle. In certain embodiments, a rAAV particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or AAV-2i8, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired). In certain embodiments, a rAAV2 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV2 particle, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired). 28 4898-4878-0311, v.1 In certain embodiments, a rAAV9 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV2 particle, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired). A rAAV particle can comprise an ITR having any suitable number of “GAGC” repeats. In certain embodiments an ITR of an AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR comprising three “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR which has less than four “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR which has more than four “GAGC” repeats. In certain embodiments an ITR of a rAAV2 particle comprises a Rep binding site wherein the fourth nucleotide in the first two “GAGC” repeats is a C rather than a T. Exemplary suitable length of DNA can be incorporated in rAAV vectors for packaging / encapsidation into a rAAV particle can about 5 kilobases (kb) or less. In particular, embodiments, length of DNA is less than about 5kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb. rAAV vectors that include a nucleic acid sequence that directs the expression of an RNAi or polypeptide can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transduction-competent AAV particles and propagated using an AAV viral packaging system. A transduction-competent AAV particle is capable of binding to and entering a mammalian cell and subsequently delivering a nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, an intact rAAV particle that is transduction-competent is configured to transduce a mammalian cell. A rAAV particle configured to transduce a mammalian cell is often not replication competent, and requires additional protein machinery to self-replicate. Thus, a rAAV particle that is configured to transduce a mammalian cell is engineered to bind and enter a mammalian cell and deliver a nucleic acid to the cell, wherein the nucleic acid for delivery is often positioned between a pair of AAV ITRs in the rAAV genome. 29 4898-4878-0311, v.1 Suitable host cells for producing transduction-competent AAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells that can be, or have been, used as recipients of a heterologous rAAV vectors. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. In certain embodiments a modified human embryonic kidney cell line (e.g., HEK293), which is transformed with adenovirus type-5 DNA fragments, and expresses the adenoviral E1a and E1b genes is used to generate recombinant AAV particles. The modified HEK293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV particles. Methods of generating high titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particle can be made as set forth in Wright, 2008 and Wright, 2009. In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. A number of other vectors are known which encode Rep and / or Cap expression products. The AAV may comprise a modified AAV capsid protein derived from an AAV1 capsid protein (e.g., SEQ ID NO: 7), wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, the linker sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 8, which shows the targeting peptide insertion after position 590 as SSAX7AS, where the leading SSA and the trailing AS are linker sequences and X7 represents the targeting peptide. The wild- type AAV1 capsid protein sequence is provided in SEQ ID NO: 8. In some aspects, the 30 4898-4878-0311, v.1 modified AAV1 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 8. In some aspects, the targeting peptide is one of the peptides of LGGSAAR (SEQ ID NO: 13); IDVGSAD (SEQ ID NO: 14); FAAMGSL (SEQ ID NO: 15); RDATRSS (SEQ ID NO: 16); RPGREAS (SEQ ID NO: 17); TAPKSLK (SEQ ID NO: 18); DKTRAGS (SEQ ID NO: 19); or NSVRPLT (SEQ ID NO: 20). SEQ ID NO: 7: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQ PLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWA LPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPK RLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRL MNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDN NNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNV MITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVE IEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL SEQ ID NO: 8 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQ PLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWA LPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPK RLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRL MNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDN NNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNV MITDEEEIKATNPVATERFGTVAVNFQSSSTDSSAXXXXXXXASPATGDVHAMGALPGMVWQ DRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASF ITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYL TRPL The AVV may comprise a modified AAV capsid protein derived from an AAV2 capsid protein (e.g., SEQ ID NO: 9), wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO: 10, which shows the targeting peptide insertion after position 587 as AAAX7AA, where the leading AAA and the trailing AA are linker sequences and X7represents the targeting peptide. In 31 4898-4878-0311, v.1 some aspects, the modified AAV2 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 10. In some aspects, the targeting peptide is one of the peptides of AAKVAAP (SEQ ID NO: 21); KAGGSQG (SEQ ID NO: 22); PPYAVMV (SEQ ID NO: 23); HDGGASR (SEQ ID NO: 24); KAQGVGG (SEQ ID NO: 25); or EAQSHPR (SEQ ID NO: 26). SEQ ID NO: 9 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKG EPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR VLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWA LPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLM NPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNN NSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAK IPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEI EWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 10 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKG EPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR VLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWA LPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLM NPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNN NSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNAAAXXXXXXXAARQAATADVNTQGVLPGMVWQD RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFI TQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLT RNL The AAV may comprise a modified AAV capsid protein derived from an AAV9 capsid protein (e.g., SEQ ID NO: 11), wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, the linker sequences are AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO: 12, which shows the targeting peptide insertion after position 588 as AAAX7AS, where the leading AAA and the trailing AS are linker sequences and X7represents the targeting peptide. In 32 4898-4878-0311, v.1 some aspects, the modified AAV9 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 12. In some aspects, the targeting peptide is one of the peptides of RGDLQWV (SEQ ID NO: 27); RSGVGSA (SEQ ID NO: 28); SRGSGPS (SEQ ID NO: 29); MMGRPGR (SEQ ID NO: 30); KGGGFHG (SEQ ID NO: 31); AGVKPGR (SEQ ID NO: 32); or RGGGVYG (SEQ ID NO: 33). SEQ ID NO: 11 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR LLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQ PIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWA LPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRP KRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDR LMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQN NNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKV MITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWA KIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVE IEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL SEQ ID NO: 12 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR LLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQ PIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWA LPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRP KRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDR LMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQN NNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKV MITNEEEIKTTNPVATESYGQVATNHQSAQAAAXXXXXXXASAQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSF ITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYL TRNL IV. Methods of Administration Provided herein are methods for inducing expression of a transgene in an auditory hair cell of the inner ear. In some of these embodiments, the subject has a hearing disorder, and the transgene is delivered in a therapeutically effective amount. In some embodiments, the AAV vector transduces at least about 70% of cells of the inner ear; the AVV targets inner and outer hair cells with at least about 70%, 80%, 90%, 95% or greater efficiency, even as high as 100% efficiency. In some embodiments, the cell is a cell of the vestibular system, e.g., a hair cell of the utricle, or a cell in an ampulla of a lateral 33 4898-4878-0311, v.1 semicircular canal, or a hair cell in a cupula. In some embodiments wherein the cell is a cell of the vestibular system, the subject has a disorder of the vestibular system, and the transgene is delivered in a therapeutically effective amount. The cochlea has two types of hair cells: Inner hair cells (IHCs) convert the mechanical stimulus of sound vibration into a neural signal transmitted by type I spiral ganglion neurons to the brain. Outer hair cells (OHCs) connect only to poorly defined type II neurons; their main function is to amplify the vibration produced by sound by as much as 60 decibels (dB) in a frequency-specific manner, and they are essential for frequency discrimination (important in speech perception). Most deafness genes known to affect hair cell function are expressed in both cell types, so in general, a useful gene therapy strategy should target both IHCs and OHCs. Cells, including hair cells, in the vestibular system, e.g., in the semicircular ducts (horizontal, anterior and posterior) or two otolith organs (saccule and utricle), are essential for our sense of balance and for coordinating eye movements; they are often affected in hereditary deafness so gene therapies should target them as well. The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells. These compositions can be used to treat a condition associated with loss of hearing or vestibular dysfunction, wherein the condition is caused by a genetic defect or is ameliorated by genetic therapy. Thus, in some embodiments, the methods described herein are used to treat a condition listed in Table A, using the corresponding sequence listing in Table A, in a subject in need thereof. Examples include certain forms of Usher syndrome (deafness associated with blindness and in some forms vestibular dysfunction). In one example, the hearing loss is presbycusis. In another example, the hearing loss is high-frequency hearing loss. The high-frequency hearing loss is at 2 kHz and above. In yet another example, the hearing loss is due to ototoxicity, noise induced hearing loss, viral infections of the inner ear, autoimmune inner ear diseases, genetic hearing losses, inner ear barotrauma; physical trauma, or surgical trauma; or inflammation. The ototoxicity 34 4898-4878-0311, v.1 results from cisplatin treatment of the subject suffering from cancer. In one example, the hereditary hearing loss is Usher’s I syndrome, Usher’s II syndrome or Usher’s III syndrome. In one example, the impaired balance is in a subject who is aging. In another example, the impaired vestibular function is result of vestibular organ degeneration. The vestibular organ regeneration is due to ototoxicity, viral infections of the inner ear, autoimmune inner ear diseases, genetic vestibular losses, inner ear barotraumas; or physical trauma, or surgical trauma. For example, genetically based hearing loss is a significant problem with few therapeutic options other than cochlear implants. Inherited hearing problems are often due to single gene defects. Prelingual deafness is diagnosed in 1 / 500 infants, of which about 50% have a genetic etiology. Usher syndrome, which is associated with a number of different clinical subtypes, each of which can be caused by a mutation in any of a number of different genes, is responsible for 3 to 6% of early childhood deafness. One of the more prevalent genetic defects, estimated to be 1-2% of all genetic deafness, occurs in the TMC1 gene. Usher syndrome is classified under three clinical subtypes (USH-1, -2 and -3) according to the severity of the symptoms. USH1 is the most severe form. Patients who are affected by USH1 suffer congenital bilateral profound sensorineural hearing loss, vestibular areflexia and pre-pubertal retinitis pigmentosa (a progressive, bilateral, symmetric degeneration of rod and cone function of the retina). Unless fitted with a cochlear implant, individuals do not typically develop the ability to generate speech. While no biological treatments currently exist for Usher patients, early re introduction of the wild-type form of the defective gene may allow for reversal of the disease. The most severe form of Usher Syndrome, USH1, is associated with defects in six genes: USH1, MY07A (myosin 7a), USH1C (harmonin), CDH23(cadherin 23), PCDH15 (protocadherin 15), SANS (sans; also known as USHIG) and CIB2 (calcium and integrin binding protein2). These genes encode proteins that are involved in hair bundle morphogenesis in the inner ear and are part of an interactome (see, for example, Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20). Harmonin resides at the center of the USH1 interactome where it binds to other Usher 1 proteins. Because of its PDZ (PSD-59 95 / Dlg / ZO- l) interaction domains, harmonin has been proposed to function as a scaffolding protein. In vitro binding studies have shown that all other known USH1 proteins bind to PDZ domains of harmonin as do two of the USH2 proteins, usherin, and VLGRl. The USHI C 35 4898-4878-0311, v.1 gene consists of 28 exons, which code for 10 alternative splice forms of harmonin, grouped into three different subclasses (a, b and c) depending on the domain composition of the protein. The three isoforms differ in the number of PDZ protein-protein interaction domains, coiled-coiled (CC) domains, and proline-serine-threonine (PST) rich domains. USH1 proteins are localized to the apex of hair cells in mechanosenosory hair bundles, which are composed of hundreds of stereocilia interconnected by numerous extracellular links. Cadherin 23 and Protocadherin 15, products of Usher genes (USHI D and USH1 E, respectively) form tip- links located at the distal end of the stereocilia. Harmonin-b binds to CDH23, PCDH15, F- actin and itself. It is found at the tips of the stereocilia near the tip-link insertion point in hair cells where it is thought to play a functional role in transduction and adaptation in hair cells. Harmonin-b is expressed during early postnatal stages but its expression diminishes around postnatal day 30 (P30) in both the cochlea and vestibule. Harmonin-a also binds to cadherin 23 and is found in the stereocilia. Recent reports reveal an additional role for harmonin-a at the synapse where it associates with Cavl .3 Ca2+ channels to limit channel availability through a ubiquitin-dependent pathway. Several mouse models for Usher syndrome have been identified or engineered over the past decade, seven of which affect harmonin. Of these, only one model, the Ush l c c.216G>A model, reproduces both auditory and retinal deficits that characterize human Usher Syndrome. Ushlc c.216G>A is a knock-in mouse model that affects expression of all conventional harmonin isoforms due a point mutation similar to the one found in a cohort of French-Acadian USHI C patients. The mutation introduces a cryptic splice site at the end of exon three of the Ushl c gene. Use of this cryptic splice site produces a frame-shifted transcript with a 35 bp deletion and results in translation of a severely truncated protein lacking PDZ, PST and CC domains. Homozygous C.216AA knock-in mice suffer from severe hearing loss at 1 month of age while heterozygous C.216GA mice do not present any abnormal phenotype. Cochlear histology in C.21 AA mice shows disorganized hair bundles, abnormal cell rows and loss of both inner and outer hair cells in middle and basal turns at P30. Over 40 distinct mutations have been identified in TMC1 that cause deafness. These are subdivided into 35 recessive mutations and 5 dominant mutations. Most of the recessive mutations cause profound, congenital hearing loss (e.g., DFNB7 / 11) though a few cause later onset, moderate to severe hearing loss. All of the dominant mutations cause 36 4898-4878-0311, v.1 progressive hearing loss (e.g., DFNA36), with onset in the mid-teen years. In particular, an AAV vector that includes an Anc80 capsid protein as described herein can be used to deliver a non-mutant (e.g., wild type) TMC1 sequence or TMC2 sequence, thereby preventing hearing loss (e.g., further hearing loss) and / or restoring hearing function. Therapeutic gene transfer to the cochlea has been considered to further improve upon the current standard of care ranging from age-related and environmentally induced hearing loss to genetic forms of deafness. More than 300 genetic loci have been linked to hereditary hearing loss with over 70 causative genes described (Parker & Bitner- Glindzicz, 2015, Arch. Dis. Childhood, 100:271 -8). Therapeutic success in these approaches relies significantly on the safe and efficient delivery of exogenous gene constructs to the relevant therapeutic cell targets in the organ of Corti (OC) in the cochlea. The OC includes two classes of sensory hair cells: IHCs, which convert mechanical information carried by sound into electrical signals transmitted to neuronal structures and OHCs which serve to amplify and tune the cochlear response, a process required for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells of the spiral limbus, which are important for the maintenance of the adjacent tectorial membrane or supporting cells, which have protective functions and can be triggered to trans-differentiate into hair cells up to an early neonatal stage. As used herein, inner ear cells refer to, without limitation, inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, vestibular ganglion neurons, and supporting cells. Supporting cells refer to cells in the ear that are not excitable, e.g., cells that are not hair cells or neurons. An example of a supporting cell is a Schwann cell. Any suitable cell or mammal can be administered or treated by a method or use described herein. Typically, a mammal in need of a method described herein is suspected of having or expressing an abnormal or aberrant protein that is associated with a disease state. Alternative, the mammalian recipient may have a condition that is amenable to gene replacement therapy. As used herein, “gene replacement therapy” refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase “condition amenable to gene replacement therapy” embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects) and acquired pathologies (i.e., a 37 4898-4878-0311, v.1 pathological condition which is not attributable to an inborn defect). Accordingly, as used herein, the term “therapeutic agent” refers to any agent or material, which has a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” embraces both therapeutic and prophylactic molecules having nucleic acid or protein components. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In certain embodiments a mammal is a human. In certain embodiments a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like). In certain embodiments a non-rodent mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In certain embodiments a mammal can be an animal disease model, for example, animal models having or expressing an abnormal or aberrant protein that is associated with a disease state or animal models with insufficient expression of a protein, which causes a disease state. Mammals (subjects) treated by a method or composition described herein include adults (18 years or older) and children (less than 18 years of age). Adults include the elderly. Representative adults are 50 years or older. Children range in age from 1-2 years old, or from 2–4, 4–6, 6–18, 8–10, 10–12, 12–15 and 15–18 years old. Children also include infants. Infants typically range from 1–12 months of age. In certain embodiments, a method includes administering a plurality of viral particles to a mammal as set forth herein, where severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, decreased, reduced, prevented, inhibited or delayed. In certain embodiments, a method includes administering a plurality of viral particles to a mammal to treat an adverse symptom of a disease state, such as a neuro-degenerative disease. In certain embodiments, a method includes administering a plurality of viral particles to a mammal to stabilize, delay or prevent worsening, or progression, or reverse and adverse symptom of a disease state, such as a neuro-degenerative disease. In certain embodiments a method includes administering a plurality of viral particles to the central nervous system, or portion thereof as set forth herein, of a mammal 38 4898-4878-0311, v.1 and severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, are decreased, reduced, prevented, inhibited or delayed by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days. In some embodiments, a composition comprising a therapeutically effective number of virus particles containing a transgene, or containing one or more sets of different virus particles, wherein each particle in a set can contain the same type of transgene, but wherein each set of particles contains a different type of transgene than in the other sets, as described herein can be delivered. Formulations according to the present invention can be used for CNS delivery via various techniques and routes including, but not limited to, intraparenchymal, intracerebral, intravetricular cerebral (ICV), intrathecal (e.g., IT-Lumbar, IT-thoracic, IT- cisterna magna) administrations and any other techniques and routes for injection directly or indirectly to the CNS and / or CSF. In some embodiments, a formulation is delivered to the CNS by administering into the cerebrospinal fluid (CSF) of a subject in need of treatment. In some embodiments, intrathecal administration is used to deliver viral particles into the CSF. As used herein, intrathecal administration (also referred to as intrathecal injection) refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like. Exemplary methods are described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of which are incorporated herein by reference. According to the present invention, viral particles may be injected at any region surrounding the spinal canal. In some embodiments, viral particles are injected into the lumbar area or the cisterna magna or intraventricularly into a cerebral ventricle space. As used herein, the term “lumbar region” or “lumbar area” refers to the area between the third and fourth lumbar (lower back) vertebrae and, more inclusively, the L2-S 1 region of the spine. Typically, intrathecal injection via the lumbar region or lumber area is also referred to as “lumbar IT delivery” or “lumbar IT administration.” The term “cisterna magna” refers to 39 4898-4878-0311, v.1 the space around and below the cerebellum via the opening between the skull and the top of the spine. Typically, intrathecal injection via cisterna magna is also referred to as “cisterna magna delivery.” The term “cerebral ventricle” refers to the cavities in the brain that are continuous with the central canal of the spinal cord. As such, intrathecal administration includes any infusion into the central canal. Typically, injections via the cerebral ventricle cavities are referred to as intravetricular cerebral (ICV) delivery. Various devices may be used for intrathecal delivery according to the present invention. In some embodiments, a device for intrathecal administration contains a fluid access port (e.g., injectable port); a hollow body (e.g., catheter) having a first flow orifice in fluid communication with the fluid access port and a second flow orifice configured for insertion into spinal cord; and a securing mechanism for securing the insertion of the hollow body in the spinal cord. In various embodiments, the fluid access port comprises a reservoir. In some embodiments, the fluid access port comprises a mechanical pump (e.g., an infusion pump). In some embodiments, an implanted catheter is connected to either a reservoir (e.g., for bolus delivery), or an infusion pump. The fluid access port may be implanted or external In some embodiments, intrathecal administration may be performed by either lumbar puncture (i.e., slow bolus) or via a port-catheter delivery system (i.e., infusion or bolus). In some embodiments, the catheter is inserted between the laminae of the lumbar vertebrae and the tip is threaded up the thecal space to the desired level (generally L3-L4). A single dose volume suitable for intrathecal administration is typically small. Typically, intrathecal delivery according to the present invention maintains the balance of the composition of the CSF as well as the intracranial pressure of the subject. In some embodiments, intrathecal delivery is performed absent the corresponding removal of CSF from a subject. In some embodiments, a suitable single dose volume may be e.g., less than about 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5 ml. In some embodiments, a suitable single dose volume may be about 0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3 ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal delivery according to the present invention involves a step of removing a desired amount of CSF first. In some embodiments, less than about 10 ml (e.g., less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) of CSF is first removed before IT administration. In those cases, a suitable single dose volume may be e.g., more than about 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml. 40 4898-4878-0311, v.1 Various other devices may be used to effect intrathecal administration of a therapeutic composition. For example, formulations containing desired enzymes may be given using an Ommaya reservoir which is in common use for intrathecally administering drugs for meningeal carcinomatosis (Lancet 2: 983-84, 1963). More specifically, in this method, a ventricular tube is inserted through a hole formed in the anterior horn and is connected to an Ommaya reservoir installed under the scalp, and the reservoir is subcutaneously punctured to intrathecally deliver the particular enzyme being replaced, which is injected into the reservoir. Other devices for intrathecal administration of therapeutic compositions or formulations to an individual are described in U.S. Pat. No. 6,217,552, incorporated herein by reference. Alternatively, the viral particles may be intrathecally given, for example, by a single injection, or continuous infusion. It should be understood that the dosage treatment may be in the form of a single dose administration or multiple doses. In one embodiment of the invention, the viral particles are administered by lateral cerebro ventricular injection into the brain of a subject. The injection can be made, for example, through a burr hole made in the subject’s skull. In another embodiment, the viral particles and / or other pharmaceutical formulation are administered through a surgically inserted shunt into the cerebral ventricle of a subject. For example, the injection can be made into the lateral ventricles, which are larger. In some embodiments, injection into the third and fourth smaller ventricles can also be made. In yet another embodiment, the pharmaceutical compositions used in the present invention are administered by injection into the cisterna magna, or lumbar area of a subject. V. Pharmaceutical Compositions As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Such composition, “pharmaceutically acceptable” and “physiologically acceptable” formulations and compositions can be sterile. Such pharmaceutical formulations and compositions may be used, for example in administering a viral particle or nanoparticle to a subject. 41 4898-4878-0311, v.1 Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in- oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the formulations and compositions. Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes. Pharmaceutical forms suitable for injection or infusion of viral particles or nanoparticles can include sterile aqueous solutions or dispersions which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate form should be a sterile fluid and stable under the conditions of manufacture, use and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Isotonic agents, 42 4898-4878-0311, v.1 for example, sugars, buffers or salts (e.g., sodium chloride) can be included. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Solutions or suspensions of viral particles or nanoparticles can optionally include one or more of the following components: a sterile diluent such as water for injection, saline solution, such as phosphate buffered saline (PBS), artificial CSF, a surfactants, fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), glycerin, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Pharmaceutical formulations, compositions and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20thed., Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11thed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp.253-315). Viral particles, nanoparticles, and their compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms are dependent upon the number of viral particles or nanoparticles believed necessary to produce the desired effect(s). The amount necessary can be formulated in a single dose, or can be formulated in multiple dosage units. The dose may be adjusted to a suitable viral particle or nanoparticle concentration, optionally combined with an anti- inflammatory agent, and packaged for use. 43 4898-4878-0311, v.1 In one embodiment, pharmaceutical compositions will include sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate symptoms or an adverse effect of a disease state in question or an amount sufficient to confer the desired benefit. A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Thus, for example, viral particles, nanoparticles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage. Formulations containing viral particles or nanoparticles typically contain an effective amount, the effective amount being readily determined by one skilled in the art. The viral particles or nanoparticles may typically range from about 1% to about 95% (w / w) of the composition, or even higher if suitable. The quantity to be administered depends upon factors such as the age, weight and physical condition of the mammal or the human subject considered for treatment. Effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose response curves. VI. Definitions The terms “polynucleotide,” “nucleic acid” and “transgene” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides can include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). Polynucleotides can be single 44 4898-4878-0311, v.1 stranded, double stranded, or triplex, linear or circular, and can be of any suitable length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5ʹ to 3ʹ direction. A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions. Nucleic acids can include one or more expression control or regulatory elements operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like. Expression control / regulatory elements can be obtained from the genome of any suitable organism. A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. A pol II promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and optionally other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A type 1 pol III promoter includes three cis-acting sequence elements downstream of the transcriptional start site: a) 5'sequence element (A block); b) an intermediate sequence element (I block); c) 3' sequence element (C block). A type 2 pol III promoter includes two essential cis-acting sequence elements downstream of the transcription start site: a) an A box (5' sequence element); and b) a B box (3' sequence element). A type 3 pol III promoter includes several cis-acting promoter elements upstream of the transcription start site, such as a traditional TATA box, proximal sequence element (PSE), and a distal sequence element (DSE). An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level 45 4898-4878-0311, v.1 or tissue specificity of expression. It is capable of operating in either orientation (5’->3’ or 3’- >5’), and may be capable of functioning even when positioned either upstream or downstream of the promoter. Promoters and / or enhancers may be derived in their entirety from a native gene, or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions. Non-limiting examples include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter, and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert. A “transgene” is used herein to conveniently refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes an inhibitory RNA or polypeptide or protein, and are generally heterologous with respect to naturally occurring AAV genomic sequences. 46 4898-4878-0311, v.1 The term “transduce” refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (e.g., a viral particle). Introduction of a transgene into a cell by a viral particle is can therefore be referred to as “transduction” of the cell. The transgene may or may not be integrated into genomic nucleic acid of a transduced cell. If an introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extra chromosomally, or only transiently. A “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which a transgene has been introduced. A transduced cell can be propagated, transgene transcribed and the encoded inhibitory RNA or protein expressed. For gene therapy uses and methods, a transduced cell can be in a mammal. Transgenes under control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting a suitable promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of a polypeptide in the genetically modified cell. If the gene encoding the polypeptide is under the control of an inducible promoter, delivery of the polypeptide in situ is triggered by exposing the genetically modified cell in situ to conditions for permitting transcription of the polypeptide, e.g., by intraperitoneal injection of specific inducers of the inducible promoters which control transcription of the agent. For example, in situ expression by genetically modified cells of a polypeptide encoded by a gene under the control of the metallothionein promoter, is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ. A nucleic acid / transgene is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding 47 4898-4878-0311, v.1 and RNAi or a polypeptide, or a nucleic acid directing expression of a polypeptide may include an inducible promoter, or a tissue-specific promoter for controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette. In certain embodiments, cell-type-specific or inducible promoters, enhancers and the like, are employed in the methods and uses described herein. Non-limiting examples of cell-type-specific promoters include those isolated from the genes from TMC1, TMC2, Espin, PCDH15, PTPRQ, TMHS (LHFPL5), MYO1A. Non-limiting examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia and IFN. In certain embodiments, an expression control element comprises a CMV enhancer. In certain embodiments, an expression control element comprises a beta actin promoter. In certain embodiments, an expression control element comprises a chicken beta actin promoter. In certain embodiments, an expression control element comprises a CMV enhancer and a chicken beta actin promoter. As used herein, the terms “modify” or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, e.g., a missense or nonsense mutation. A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of a protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of a protein encoded thereby. 48 4898-4878-0311, v.1 The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptides” encoded by a “nucleic acid” or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Accordingly, in methods and uses of the invention, such polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal. Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 or more nucleotides or residues). An example of an amino acid modification is a conservative amino acid substitution or a deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (e.g., wild-type sequence). Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors or nanoparticles, to the central nervous system, such as vascular endothelial cells. Thus, for example, endothelial cells lining brain blood vessels can be targeted by the modified recombinant viral particles or nanoparticles. A recombinant virus so modified may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, a recombinant virus bearing a modified capsid protein may “target” brain vascular epithelia tissue by binding at level higher than a comparable, unmodified capsid protein. For example, a recombinant virus having a modified capsid protein may bind to brain vascular epithelia tissue at a level 50% to 100% greater than an unmodified recombinant virus. A “nucleic acid fragment” is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while 49 4898-4878-0311, v.1 ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By “fragment” or “portion” is meant a full length or less than full length of the nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein. In certain embodiments, the fragment or portion is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type). A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type). “Conservative variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein that encodes a polypeptide also describes every possible silent 50 4898-4878-0311, v.1 variation, except where otherwise noted. One of skill in the art will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid that encodes a polypeptide is implicit in each described sequence. The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%. The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. By “mechanosensation” is meant a response to a mechanical stimulus. Touch, hearing, and balance of examples of the conversion of a mechanical stimulus into a neuronal signal. Mechanosensory input is converted into a response to a mechanical stimulus through a process termed “mechanotransduction.” By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include genetic disorders characterized by a loss of function in a protein that functions in mechanosensory 51 4898-4878-0311, v.1 transduction that is expressed, for example, in the inner ear of a subject. In another embodiment, the disease is Usher Syndrome (e.g., USH1) or age-related hearing loss. In one embodiment, a disease is an auditory disorder associated with a genetic defect, such as a defect in TMC1, TMC2, MY07A, USCH1 C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHKN, CLRN1, PDZD7, USH1C (e.g., harmonin-a, b, or c). The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, inhibit, reduce, or decrease an undesired physiological change or disorder, such as the development, progression or worsening of the disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed (e.g., as determined by a genetic assay). The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. All methods and uses described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as” or “for example”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., modified nucleic acid, vector, plasmid, a recombinant vector sequence, vector genome, or viral particle) are an example of a genus of equivalent or similar features. 52 4898-4878-0311, v.1 As used herein, the forms “a”, “and,” and “the” include singular and plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of such nucleic acids, reference to “a vector” includes a plurality of such vectors, and reference to “a virus” or “AAV or rAAV particle” includes a plurality of such virions / AAV or rAAV particles. The term “about” as used herein refers to a values that is within 10% (plus or minus) of a reference value. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Accordingly, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 100, includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10, includes 9, 8, 7, etc. all the way down to the number one (1). As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of 53 4898-4878-0311, v.1 ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500- 2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500- 6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000, includes ranges of 10-20, 10-50, 30-50, 50- 100, 100-300, 100-1,000, 1,000-3,000, 2,000-4,000, 4,000-6,000, etc. VII. Kits The invention provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, and / or viral particles. A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.). Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein. Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the 54 4898-4878-0311, v.1 subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities. Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD- ROM / RAM, DVD, MP3, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH memory, hybrids and memory type cards. VIII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 – Eps8-derived splicing cassette restricts AAV payload expression to auditory hair cells CSF-mediated delivery of the top modified AAV variants yielded capsids capable of nearly complete IHC transduction across all cochlear turns, robust transduction of spiral ganglion neurons, and transduction of non-sensory supporting cells in the organ of Corti of the NHP cochlea. Additionally, using available single-cell RNA-Seq data, a hair- cell-specific splice event with 100% sequence conservation between mouse and human was tested for its ability to confer hair cell-specific payload expression (FIGS. 1A-1B). After ICV administration to mouse CSF, there was auditory hair cell-specific gene expression, and no expression throughout the non-cochlear central nervous system (FIG.1C). 55 4898-4878-0311, v.1 * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 56 4898-4878-0311, v.1

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising a first expression cassette comprising, from 5’ to 3’, (a) a minigene having an alternatively spliced exon and (b) an encoded transgene; wherein the minigene comprises, from 5’ to 3’, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3, wherein Exon 2 is the alternatively spliced exon, wherein Exon 2 is derived from Exon 18C of the human Esp8 gene, and wherein Exon 2 comprises translation initiation regulatory sequences.

2. The nucleic acid molecule of claim 1, wherein Exon 2 comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

2.

3. The nucleic acid molecule of claim 1 or 2, wherein Exon 1 is derived from Exon 18 of the human Esp8 gene.

4. The nucleic acid molecule of any one of claims 1-3, wherein Exon 1 comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

1.

5. The nucleic acid molecule of any one of claims 1-4, wherein Exon 3 is derived from Exon 19 of the human Esp8 gene.

6. The nucleic acid molecule of any one of claims 1-5, wherein Exon 3 comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

3.

7. The nucleic acid molecule of any one of claims 1-6, wherein Intron 1 comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

4.

8. The nucleic acid molecule of any one of claims 1-7, wherein Intron 2 comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

5. 57 4898-4878-0311, v.

19. The nucleic acid molecule of any one of claims 1-8, wherein the minigene comprises or consists of a sequence having at least 90%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:

6.

10. The nucleic acid molecule of any one of claims 1-9, wherein the minigene comprises or consists of a sequence according to SEQ ID NO:

6.

11. The nucleic acid molecule of any one of claims 1-10, wherein the encoded transgene is in frame with the translation initiation regulatory sequence in Exon 2.

12. The nucleic acid molecule of any one of claims 1-11, wherein the expression of the encoded gene does not require the co-expression of any exogenous regulatory protein.

13. The nucleic acid molecule of any one of claims 1-12, wherein the encoded gene encodes a therapeutic protein, a Cas9 protein, or a transactivator protein.

14. The nucleic acid molecule of claim 13, wherein the therapeutic protein is a protein whose deficiency is associated with hearing loss.

15. The nucleic acid molecule of any one of claims 1-14, wherein the minigene and the encoded gene are separated by a cleavable peptide.

16. The nucleic acid molecule of any one of claims 1-15, wherein the first expression cassette is operably linked to a first promoter.

17. The nucleic acid molecule of claim 16, wherein the first promoter is a constitutive promoter.

18. The nucleic acid molecule of claim 17, wherein the first promoter is a Rous sarcoma virus (RSV) promoter, the phosphoglycerate kinase (PGK) promoter, a JeT promoter, a CBA promoter, a synapsin promoter, or the minimal cytomegalovirus (mCMV) promoter.

19. The nucleic acid molecule of claim 16, wherein the therapeutic transgene is operably linked to a cochlea-specific promoter.

20. The nucleic acid molecule of claim 19, wherein the cochlea-specific promoter is an auditory hair cell-specific or -selective promoter. 58 4898-4878-0311, v.

121. The nucleic acid molecule of any one of claims 1-20, further comprising a second expression cassette.

22. The nucleic acid molecule of claim 21, wherein the second expression cassette comprises a nucleic acid sequence encoding a guide RNA operably linked to a second promoter.

23. The nucleic acid molecule of claim 21, wherein the second expression cassette comprises a nucleic acid sequence encoding a therapeutic protein, an inhibitory RNA, or a Cas9 protein, wherein the nucleic acid sequence is operably linked to a second promoter, wherein the second promoter is activated by the transactivator encoded by the first expression cassette.

24. A cell comprising the nucleic acid molecule of any one of claims 1-23.

25. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein and nucleic acid molecule of any one of claims 1-24.

26. The rAAV of claim 25, wherein the rAAV comprises a modified capsid protein.

27. The rAAV of claim 26, wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide is three to ten amino acids in length.

28. The rAAV of claim 26 or 27, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

29. The rAAV of claim 28, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

30. The rAAV of claim 29, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

31. The rAAV of claim 30, wherein the linker sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. 59 4898-4878-0311, v.

132. The rAAV of any one of claims 29-31, wherein the targeting peptide is selected from the group consisting of LGGSAAR (SEQ ID NO: 13); IDVGSAD (SEQ ID NO: 14); FAAMGSL (SEQ ID NO: 15); RDATRSS (SEQ ID NO: 16); RPGREAS (SEQ ID NO: 17); TAPKSLK (SEQ ID NO: 18); DKTRAGS (SEQ ID NO: 19); or NSVRPLT (SEQ ID NO: 20).

33. The rAAV of claim 28, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

34. The rAAV of claim 33, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

35. The rAAV of claim 34, wherein the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

36. The rAAV of any one of claims 33-35, wherein the targeting peptide is selected from the group consisting of AAKVAAP (SEQ ID NO: 21); KAGGSQG (SEQ ID NO: 22); PPYAVMV (SEQ ID NO: 23); HDGGASR (SEQ ID NO: 24); KAQGVGG (SEQ ID NO: 25); or EAQSHPR (SEQ ID NO: 26).

37. The rAAV of claim 28, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

38. The rAAV of claim 37, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

39. The rAAV of claim 38, wherein the linker sequences are AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

40. The rAAV of any one of claims 37-39, wherein the targeting peptide is selected from the group consisting of RGDLQWV (SEQ ID NO: 27); RSGVGSA (SEQ ID NO: 28); SRGSGPS (SEQ ID NO: 29); MMGRPGR (SEQ ID NO: 30); KGGGFHG (SEQ ID NO: 31); AGVKPGR (SEQ ID NO: 32); or RGGGVYG (SEQ ID NO: 33). 60 4898-4878-0311, v.

141. The rAAV of any one of claims 25-40, wherein the targeting peptide is seven amino acids in length.

42. A pharmaceutical composition comprising the rAAV of any one of claims 25-41 and a pharmaceutically acceptable carrier.

43. A method of administering the encoded transgene to a patient in need thereof, the method comprising administering the nucleic acid of any one of claims 1-23 or the rAAV of any one of claims 25-41 or the pharmaceutical composition of claim 42 to the patient.

44. The method of claim 43, wherein the administration is to the cerebrospinal fluid of the patient.

45. The method of claim 43 or 44, wherein the administration comprises intracerebroventricular administration.

46. The method of any one of claims 43-45, wherein the method delivers the therapeutic transgene to the spiral organ of Corti.

47. The method of any one of claims 43-46, wherein the method delivers the therapeutic transgene to a cell of the inner ear.

48. The method of claim 47, wherein the cell in the inner ear is an auditory hair cell.

49. The method of any one of claims 43-48, wherein the method treats or prevents hearing loss.

50. The method of any one of claims 43-49, wherein the subject has a hearing disorder, and the molecular therapeutic is delivered in a therapeutically effective amount.

51. The method of any one of claims 43-50, wherein the method reverses or prevents hearing loss.

52. The method of claim 51, wherein the hearing loss is partial hearing loss or complete deafness.

53. The method of any one of claims 43-52, wherein the method treats or prevents hearing loss in a patient. 61 4898-4878-0311, v.

154. The method of any one of claims 43-53, wherein the method treats hereditary hearing loss in the patient.

55. The method of any one of claims 43-54, wherein the patient is human.

56. A method of treating genetic hearing loss or for inducing auditory hair cell regeneration, the method comprising administering to a patient in need thereof a nucleic acid molecule comprising a first expression cassette comprising, from 5’ to 3’, (a) a minigene having an alternatively spliced exon that is only included in a cochlear cell type and (b) an encoded therapeutic transgene, such that the encoded therapeutic transgene is only expressed in the cochlear cell.

57. The method of claim 56, wherein the minigene comprises, from 5’ to 3’, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3, wherein Exon 2 is the alternatively spliced exon that is only included in a cochlear cell type, and wherein Exon 2 comprises translation initiation regulatory sequences.

58. The method of claim 57, wherein the encoded therapeutic transgene is in frame with the translation initiation regulatory sequence in Exon 2.

59. The method of any one of claims 56-58, wherein the expression of the encoded therapeutic transgene does not require the co-expression of any exogenous regulatory protein.

60. The method of any one of claims 56-59, wherein the encoded therapeutic transgene encodes a therapeutic protein, a Cas9 protein, or a transactivator protein.

61. The method of claim 60, wherein the therapeutic protein is a protein whose deficiency is associated with hearing loss.

62. The method of any one of claims 56-61, wherein the first expression cassette is operably linked to a first promoter.

63. The method of claim 62, wherein the first promoter is a constitutive promoter.

64. The method of claim 62, wherein the first promoter is a Rous sarcoma virus (RSV) promoter, the phosphoglycerate kinase (PGK) promoter, a JeT promoter, a CBA promoter, a synapsin promoter, or the minimal cytomegalovirus (mCMV) promoter. 62 4898-4878-0311, v.

165. The method of claim 62, wherein the therapeutic transgene is operably linked to a cochlea-specific promoter.

66. The method of claim 65, wherein the cochlea-specific promoter is an auditory hair cell-specific or -selective promoter.

67. The method of any one of claims 56-66, wherein the nucleic acid molecule is comprised in a recombinant adeno-associated virus (rAAV).

68. The method of claim 67, wherein the rAAV comprises a modified capsid protein.

69. The method of claim 68, wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide is three to ten amino acids in length.

70. The method of claim 68 or 69, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

71. The method of claim 70, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

72. The method of claim 71, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

73. The method of claim 72, wherein the linker sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

74. The method of any one of claims 71-73, wherein the targeting peptide is selected from the group consisting of LGGSAAR (SEQ ID NO: 13); IDVGSAD (SEQ ID NO: 14); FAAMGSL (SEQ ID NO: 15); RDATRSS (SEQ ID NO: 16); RPGREAS (SEQ ID NO: 17); TAPKSLK (SEQ ID NO: 18); DKTRAGS (SEQ ID NO: 19); or NSVRPLT (SEQ ID NO: 20).

75. The method of claim 70, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. 63 4898-4878-0311, v.

176. The method of claim 75, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

77. The method of claim 76, wherein the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

78. The method of any one of claims 75-77, wherein the targeting peptide is selected from the group consisting of AAKVAAP (SEQ ID NO: 21); KAGGSQG (SEQ ID NO: 22); PPYAVMV (SEQ ID NO: 23); HDGGASR (SEQ ID NO: 24); KAQGVGG (SEQ ID NO: 25); or EAQSHPR (SEQ ID NO: 26).

79. The method of claim 70, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

80. The method of claim 79, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.

81. The method of claim 80, wherein the linker sequences are AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

82. The method of any one of claims 79-81, wherein the targeting peptide is selected from the group consisting of RGDLQWV (SEQ ID NO: 27); RSGVGSA (SEQ ID NO: 28); SRGSGPS (SEQ ID NO: 29); MMGRPGR (SEQ ID NO: 30); KGGGFHG (SEQ ID NO: 31); AGVKPGR (SEQ ID NO: 32); or RGGGVYG (SEQ ID NO: 33). 64 4898-4878-0311, v.1

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