Adeno-associated viral vectors for transducing the cochlea

Abstract

Provided herein is a composition and method for delivering molecular therapeutics to the cochlea of ​​a subject.The method comprises administering adeno-associated virus (AAV) to the cerebrospinal fluid of a subject.The AAV encodes a therapeutic transgene for molecular therapy.Optionally, the therapeutic transgene can be functionally linked to a cochlea-specific promoter.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 180,394, filed April 27, 2021, the entire contents of which are incorporated herein by reference.

[0002] Reference to sequence listing This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on April 20, 2022, is named CHOPP0048WO_ST25.txt, and is 67,842 bytes in size. [Background technology]

[0003] background 1. Field The present disclosure relates generally to the fields of medicine and virology. More particularly, the present disclosure relates to compositions and methods for delivering molecular therapeutics to the cochlea.

[0004] 2. Description of Related Technology The cochlear tissue of the inner ear has long been considered one of the most difficult tissues to reach for molecular therapeutic delivery. The cochlea is located within the temporal bone, the densest bone in the human body. The cochlea occupies a series of tortuous passageways that house both the cochlear membranous labyrinth and the vestibular system. These membranes are surrounded by bone except for three small openings: the round window, the oval window, and the perilymphatic duct. The round window and oval window openings are protected by membranes that are used to facilitate pressure input and fluid expansion when auditory stimuli are transmitted as stapes-induced pressure waves in the cochlear perilymph. A non-occluding inflow point is provided in the inner ear by the round window membrane (RWM) opening. Researchers and clinicians have exploited this entry point as a delivery route for therapeutic agents, including cochlear implants and gene therapy vectors.

[0005] Much work has been done to evaluate the feasibility of many variations of this approach in the context of lipid and viral vector delivery for gene therapy. Numerous viral vectors have been evaluated for their ability to transduce cochlear cell types from the RWM inflow point in mice and NHPs. Non-invasive approaches have been attempted that avoid puncturing the RWM and instead rely on vector particle diffusion across intact or semi-intact membranes. Additional methods include canalostomy and cochleostomy, which create new holes to reach the cochlea [1]. With these methods, holes are made directly in the semicircular canals and the cochlea, respectively. Vectors are then delivered through the openings created. Hybrid methods have also been utilized. For example, the RWM+canalostomy approach has been developed that leverages fluid inflow and outflow to distribute vector particles throughout the cochlea [2]. All of the above approaches have been attempted with a wide variety of vector types, including many classical AAV capsid serotypes and more recently developed AAV capsid variants [3, 4].

[0006] Relatively little attention has been paid to the perilymphatic duct as a possible route for AAV vector delivery. It is arguably difficult to reach, and thus requires access to the intracranial space. Furthermore, the volume of CSF is considerably larger than that of the perilymph, and the brain occupying this space could take up the vector, possibly reducing the viral load reaching the cochlea and creating a greater safety risk than hearing loss. However, if these risks can be mitigated by thoughtful treatment design, and if CSF-based delivery allows for robust cochlear transduction, CSF delivery may reduce surgical risks and unintended surgical damage to cochlear tissue. Gene transfer via CSF was first observed in guinea pigs after administration of adenoviral vectors to contralateral ears [5]. In this study, systemic delivery of 25 μL of adenoviral vector was found to transduce both cochleae. However, transduction was limited to mesothelial cells close to the opening of the perilymphatic duct.

[0007] There is a need for non-traumatic, non-surgical methods for delivering molecular and gene therapy tools to cochlear sensory and supporting cells. Summary of the Invention

[0008] overview Provided herein are compositions and methods for transducing therapeutically effective amounts of sensory cells and supporting cells in both cochleae.These compositions and methods are based on the unexpected discovery that when multiple AAV vectors are introduced into CSF, they transduce inner hair cells and supporting cells bilaterally throughout all turns of the cochlea.These surprising discoveries demonstrate that robust gene transfer into the cochlea can be achieved through CSF and perilymphatic duct.

[0009] In one aspect, provided herein is a method for delivering therapeutic transgene to the cochlea of ​​a subject, comprising administering a modified adeno-associated virus (AAV) encoding therapeutic transgene to the cerebrospinal fluid of a subject.In some aspects, the therapeutic transgene treats or prevents hearing loss or vestibular disorder when expressed in the cells of the cochlea.In some aspects, the modified AAV is the modified AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.

[0010] In some aspects, the therapeutic transgene is functionally linked to a promoter. In some aspects, the promoter is homologous to the therapeutic transgene, in other words, the promoter may be a promoter that is endogenously functionally linked to the relevant gene. In some aspects, the promoter is heterologous to the therapeutic transgene. In some aspects, the promoter is a cochlea-specific promoter. The cochlea-specific promoter may be a hair cell-specific promoter or a supporting cell-specific promoter (e.g., GJB2 promoter).

[0011] In some aspects, the modified AAV comprises a modified capsid protein. The modified capsid protein may comprise a targeting peptide that may be 3-10 amino acids in length. In some aspects, the targeting peptide is 7 amino acids in length. The modified AAV capsid protein may be a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0012] In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (e.g., SEQ ID NO:164), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is SSA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide. In some aspects, the modified AAV1 capsid protein has a sequence at least 95% identical to SEQ ID NO:167. In some aspects, the targeting peptide is one of the peptides shown in FIG. 6. In some aspects, the targeting peptide is one of the peptides of SEQ ID NOs:1-44, 150, and 151. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:39, 150, and 151. In some aspects, the targeting peptide is SEQ ID NO:39. In some aspects, the targeting peptide is SEQ ID NO:150. In some aspects, the targeting peptide is SEQ ID NO:151.

[0013] In some aspects, the modified AAV capsid protein is derived from an AAV2 capsid protein (e.g., SEQ ID NO:165), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide. In some aspects, the modified AAV2 capsid protein has a sequence at least 95% identical to SEQ ID NO:168. In some aspects, the targeting peptide is one of the peptides shown in FIG. 8. In some aspects, the targeting peptide is one of the peptides of SEQ ID NOs:45-100, 152, and 154. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:84, 152, and 154. In some aspects, the targeting peptide is SEQ ID NO:84. In some aspects, the targeting peptide is SEQ ID NO:152. In some aspects, the targeting peptide is SEQ ID NO:154.

[0014] In some aspects, the modified AAV capsid protein is derived from an AAV9 capsid protein (e.g., SEQ ID NO:166), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide. In some aspects, the modified AAV9 capsid protein has a sequence at least 95% identical to SEQ ID NO:169. In some aspects, the targeting peptide is one of the peptides shown in FIG. 10. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:101-149, 153, and 155. In some aspects, the targeting peptide is SEQ ID NO:153. In some aspects, the targeting peptide is SEQ ID NO:155.

[0015] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, or AAV9. In certain aspects, the targeting peptide is inserted into position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. In an exemplary modified AAV1 capsid protein, the targeting peptide is inserted after position 590 as SSAX7AS, with the first SSA and the second AS being linker sequences, and X7 representing the targeting peptide. In an exemplary modified AAV2 capsid protein sequence, the targeting peptide is inserted after position 587 as AAAX7AA, with the first AAA and the second AA being linker sequences, and X7 representing the targeting peptide. In an exemplary modified AAV9 capsid protein sequence, position 588 is followed by a targeting peptide insertion as AAAX7AS, where the leading AAA and trailing AS are linker sequences and X7 represents the targeting peptide.

[0016] In some aspects, the therapeutic transgene encodes an siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system.

[0017] In some aspects, the administration is to the cisterna magna, intraventricular space, ventricle, subarachnoid space, and / or intrathecal space. In some aspects, the administration is via an intraventricular delivery route. In some aspects, the delivery is to the cerebrospinal fluid. In some aspects, the cochlear therapeutic delivery is facilitated by diffusing the therapeutic particles through the perilymphatic duct into the perilymph-filled scala vestibuli and scala tympani of the cochlea. In some aspects, the method delivers the therapeutic transgene to a cell of the inner ear. In some aspects, the cell in the inner ear is selected from the group consisting of a spiral ganglion neuron, a vestibular hair cell, a vestibular ganglion neuron, a supporting cell, and a cell in the stria vascularis. In some aspects, the cell is a hair cell of the cochlea or the vestibular system. In some aspects, the cell of the vestibular system is a hair cell of the utricle, or a cell in the ampulla of the lateral semicircular canal, or a hair cell in the apex. In some aspects, the cells are cochlear inner hair cells or cochlear outer hair cells, hi some aspects, the therapeutic transgene is delivered to at least 80% of the inner hair cells and / or at least 80% of the outer hair cells.

[0018] In some aspects, the subject has hearing impairment, and the therapeutic transgene is delivered in a therapeutically effective amount.In some aspects, the subject is at risk of exposure to harmful auditory stimuli.In some aspects, the administration reverses or prevents hearing loss.In some aspects, the method treats genetic hearing loss in the subject.In some aspects, the hearing loss is partial hearing loss or complete hearing loss.

[0019] In some aspects, 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. In some aspects, the method treats or prevents a balance disorder or vestibular dysfunction in a subject.

[0020] In some aspects, multiple viral particles are administered. The virus is about 1×10 6 ~Approx. 1×10 18 The virus is administered at a dose of about 1×10 vector genomes / kilogram (vg / kg). In some aspects, the virus is administered at a dose of about 1×10 virus / kg patient. 7 ~1×10 17 , about 1×10 8 ~1×10 16 , about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , about 1×10 12 ~×10 13 , or approximately 1 × 10 13 ~1×10 14 vg. In some aspects, the subject is a human.

[0021] In one aspect, provided herein is a modified adeno-associated virus (AAV), which comprises a therapeutic transgene functionally linked to a cochlear-specific promoter.In some aspects, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.In some aspects, the therapeutic transgene treats or prevents hearing loss or vestibular disorder.

[0022] In some aspects, the therapeutic transgene is functionally linked to a promoter. In some aspects, the promoter is homologous to the therapeutic transgene, in other words, the promoter may be a promoter that is endogenously functionally linked to the relevant gene. In some aspects, the promoter is heterologous to the therapeutic transgene. In some aspects, the promoter is a cochlea-specific promoter. The cochlea-specific promoter may be a hair cell-specific promoter or a supporting cell-specific promoter (e.g., GJB2 promoter).

[0023] In some aspects, the modified AAV comprises a modified capsid protein. The modified capsid protein may comprise a targeting peptide that may be 3-10 amino acids in length. In some aspects, the targeting peptide is 7 amino acids in length. The modified AAV capsid protein may be a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0024] In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (e.g., SEQ ID NO:164), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is SSA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide. In some aspects, the targeting peptide is one of the peptides shown in Figure 6. In some aspects, the targeting peptide is one of the peptides of SEQ ID NOs:1-44, 150, and 151. In some aspects, the targeting peptide is one of the peptides of SEQ ID NOs:39, 150, and 151. In some aspects, the targeting peptide is SEQ ID NO:39. In some aspects, the targeting peptide is SEQ ID NO: 150. In some aspects, the targeting peptide is SEQ ID NO: 151.

[0025] In some aspects, the modified AAV capsid protein is derived from an AAV2 capsid protein (e.g., SEQ ID NO:165), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide. In some aspects, the targeting peptide is one of the peptides shown in FIG. 8. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:45-100, 152, and 154. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:84, 152, and 154. In some aspects, the targeting peptide is SEQ ID NO:84. In some aspects, the targeting peptide is SEQ ID NO: 152. In some aspects, the targeting peptide is SEQ ID NO: 154.

[0026] In some aspects, the modified AAV capsid protein is derived from an AAV9 capsid protein (e.g., SEQ ID NO:166), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide. In some aspects, the targeting peptide is one of the peptides shown in FIG. 10. In some aspects, the targeting peptide is one of the peptides of SEQ ID NO:101-149, 153, and 155. In some aspects, the targeting peptide is SEQ ID NO:153. In some aspects, the targeting peptide is SEQ ID NO:155.

[0027] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. In certain aspects, the targeting peptide is inserted into position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. In an exemplary modified AAV1 capsid protein, the targeting peptide insertion is as SSAX7AS after position 590, with the first SSA and the second AS being linker sequences, and X7 representing the targeting peptide. In an exemplary modified AAV2 capsid protein sequence, the targeting peptide insertion is as AAAX7AA after position 587, with the first AAA and the second AA being linker sequences, and X7 representing the targeting peptide. In an exemplary modified AAV9 capsid protein sequence, position 588 is followed by a targeting peptide insertion as AAAX7AS, where the leading AAA and trailing AS are linker sequences and X7 represents the targeting peptide.

[0028] In some aspects, the therapeutic transgene encodes an siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system.

[0029] In one aspect, provided herein is a pharmaceutical composition comprising a modified AAV of any one of the present aspects and a pharma- ceutically acceptable carrier.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of example 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]

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the 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.

[0032] [Figure 1] Temporal bone of a rhesus monkey injected intracerebroventricularly (ICV) with AAV9. When the temporal bone was dissected and the bone covering the apical turn of the cochlea was removed, strong fluorescence was observed from the transduced cochlear hair cells and supporting cells. [Diagram 2] Under epifluorescence microscopy, tiled images of whole tissue preparations of rhesus cochleae from ICV AAV9-injected animals were collected. The preparation shows the organ of Corti spanning the entire length of the cochlea from the apical to the basal turns (apical, middle, and base). Robust inner hair cell and supporting cell transduction was observed in the apical and middle turns, whereas sparse IHC transduction was observed at the base. Cells transduced with AAV9 were observed throughout the entire turns of the cochlea, suggesting that all frequency-specific regions are targeted to some degree. [Diagram 3] Mouse temporal bone from an animal injected ICV with an AAV2 variant. In this image, the bone overlying the apical turn of the cochlea has been removed. Transduced tissues include the facial nerve, genticulate ganglion, and inner hair cells of the cochlea and vestibular nerve. [Figure 4] Dissecting microscope images showing the extent and specificity of transduction of inner hair cells following ICV injection of AAV2 variants across the apex and base of the mouse cochlea. [Diagram 5]Images showing transduction of inner hair cells (IHC) in mouse cochleae transduced with AAV2 variants delivered by intracerebroventricular (ICV) injection. Remarkably strong and specific IHC transduction was observed reaching cells across the apex and base of the mouse cochlea (one less turn than rhesus monkey). Here we show representative images of cochlear regions from the apex and base. Transduction was nearly >90% from the base to the mid-apex of the cochlea. Interestingly, in the rather apical tip we observed less transduction. However, this was restricted to a very small area. This level of transduction of inner hair cells was therapeutically relevant and completely unexpected from ICV brain injection into CSF. [Figure 6] The figure illustrates results from an AAV directed evolution experiment. A library of barcoded AAV1 variants was introduced into the cochlea by direct cochlear injection delivered by a RWM+ fenestration approach. Multiple injections were performed to enrich for functional capsids. Round 1 shows hits obtained after the first round of in vivo enrichment. Round 2 (DNA and RNA) shows hits obtained after the second round of in vivo enrichment. Several sequences were strongly enriched in cochlear and / or vestibular tissues. Sequences shown for the cochlea are, from top to bottom, SEQ ID NOs: 1-40. The sequences shown for vestibular tissue are, from top to bottom, SEQ ID NOs: 41, 6, 7, 3, 42, 43, 8, 12, 1, 5, 44, 4, 13, 19, 15-17, 11, 28, 20, 26, 18, 30, 25, 22, 23, 21, 31, 24, 29, 32, 40, 37, 27, 36, 34, 38, 33, 39, and 35. From among the AAV1-derived capsid candidates, capsid candidate LGGSAAR (SEQ ID NO:39) was selected due to its strong DNA and RNA performance in both the cochlea and pooled vestibular tissue. [Figure 7]Correlation of AAV1 capsid variant detection for DNA and RNA hits between cochlear and vestibular tissues. DNA capsid hits were highly correlated between the two tissues, whereas RNA hits were variable, with a low number of highly correlated capsids. [Figure 8] The figure illustrates results from an AAV directed evolution experiment. A library of barcoded AAV2 variants was introduced into the cochlea by direct cochlear injection delivered by a RWM+ fenestration approach. Multiple injections were performed to enrich for functional capsids. Round 1 shows hits obtained after the first round of in vivo enrichment. Round 2 (DNA and RNA) shows hits obtained after the second round of in vivo enrichment. Several sequences were strongly enriched in cochlear and / or vestibular tissues. Sequences shown for the cochlea are, from top to bottom, SEQ ID NOs: 45-84. The sequences shown for vestibular tissue are, from top to bottom, SEQ ID NOs: 85, 86, 69, 87-91, 56, 92, 93, 62, 94, 66, 68, 95, 96, 73, 49, 51, 97-99, 70, 65, 47, 77, 71, 75, 76, 100, 78, 80, 63, 82, 79, 81, 72, 83, and 84. Capsid candidate KAGGSQG (SEQ ID NO:84) was selected due to its strong DNA and RNA performance in the cochlea, despite its low RNA performance in vestibular tissue. [Figure 9] Correlation of AAV2 capsid variant detection for DNA and RNA hits between cochlear and vestibular tissues. DNA capsid hits were highly correlated between the two tissues, whereas RNA hits were variable, with a low number of highly correlated capsids. [Figure 10]The figure illustrates results from an AAV directed evolution experiment. A library of barcoded AAV9 variants was introduced into the cochlea by direct cochlear injection delivered by a RWM+ fenestration approach. Multiple injections were performed to enrich for functional capsids. Round 1 shows hits obtained after the first round of in vivo enrichment. Round 2 (DNA and RNA) shows hits obtained after the second round of in vivo enrichment. Several sequences were strongly enriched in cochlear and / or vestibular tissues. Sequences shown for the cochlea are, from top to bottom, SEQ ID NOs:101-140. Sequences shown for vestibular tissue are, from top to bottom, SEQ ID NOs: 110, 141-143, 117, 105, 144, 145, 129, 146, 147, 109, 130, 115, 114, 148, 111, 128, 113, 104, 121, 131, 102, 120, 149, 126, 127, 122, 125, 123, 124, 134, 133, 135, 132, 137, 136, and 138-140. No capsid candidates were selected from AAV9 because only a limited number of fluorescent validation positions (8) were available and other criteria were considered. [Figure 11] Correlation of AAV9 capsid variant detection for DNA and RNA hits between cochlear and vestibular tissues. DNA capsid hits were highly correlated between the two tissues, whereas RNA hits were variable, with a low number of highly correlated capsids. [Figure 12]Ranking of the average round 2 DNA UMI count and the average fold enrichment of the round 2 DNA UMI count compared to the UMI count from the input virus. Additional capsid candidates were selected from the average UMI count and the fold enrichment of the % round 2 DNA UMI count compared to the % DNA UMI count obtained from the round 2 input vector pool. The top non-normalized hit sequences were LGGSAAR (SEQ ID NO:39) for AAV1 and KAGGSQG (SEQ ID NO:84) for AAV2. The top normalized hits were IDVGSAD (SEQ ID NO:150) and FAAMGSL (SEQ ID NO:151) for AAV1, PPYAMVM (SEQ ID NO:152) for AAV2, and SRGSGPS (SEQ ID NO:153) for AAV9. [Figure 13] Figures 13A-C: Selection of intermediate capsid candidates with strong detection:input ratios. Figure 13A. Using this method, no capsid candidates were selected from AAV1. Figure 13B. Using this method, AAV2-derived candidate AAKVAAP (SEQ ID NO: 154) was selected. Figure 13C. Using this method, AAV9-derived candidate RSGVGSA (SEQ ID NO: 155) was selected. [Figure 14]14A-C: Capsid candidates selected for in vivo fluorescence validation. FIG. 14A. All capsid candidates selected for progression to fluorescence validation (from top to bottom: SEQ ID NOs: 39, 84, and 150-155) are listed with the parent serotype indicated and a description of the criteria used to select the capsid. FIG. 14B. To facilitate delivery of separate fluorescently tagged capsids, one to each ear of a non-human primate, capsids were grouped into two validation pools. Pool 1 peptide sequences from top to bottom: SEQ ID NOs: 39, 84, 150, and 152. Pool 1 DNA sequences from top to bottom: SEQ ID NOs: 156-159. Pool 2 peptide sequences from top to bottom: SEQ ID NOs: 154, 155, 151, and 153. Pool 2 DNA sequences from top to bottom: SEQ ID NOs: 160-163. FIG. 14C. Capsid candidates were individually generated to deliver fluorescent reporter expression constructs. Capsids were pooled in groups of four to create the validation pool described above. One validation pool is delivered by directing an intracochlear injection into each inner ear of a rhesus monkey. Thirty days after injection, animals are sacrificed and cochleae are harvested for histological evaluation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Provided herein is the unexpected discovery that a single intracerebroventricular injection of AAV vectors into the CSF is sufficient to transduce a therapeutically relevant number of sensory and supporting cells in both cochleae. Surprisingly, robust and widespread transduction of the cochlea was observed in rhesus monkeys. When AAV9 was delivered into the CSF by ICV injection, it bilaterally transduced inner hair cells and supporting cells throughout all turns of the cochlea. Other vectors delivered ICV to mice were found to have similar cochlear transduction capacity with various cell type specificities. AAV2-derived capsid variants delivered ICV to mice were found to have a high degree of specificity for inner hair cells, resulting in nearly complete IHC transduction in all turns except the apical tip. These surprising findings reveal that robust gene transfer into the cochlea can be achieved via the CSF and perilymphatic duct. This observation of gene transfer in adult rhesus macaques suggests that a similar ICV-based approach may be successful in humans.

[0034] In certain embodiments, the AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is shown in SEQ ID NO:164. An exemplary wild-type reference AAV2 capsid protein sequence is shown in SEQ ID NO:165. An exemplary wild-type reference AAV9 capsid protein sequence is shown in SEQ ID NO:166. In certain aspects, the targeting peptide is inserted at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. An exemplary modified AAV1 capsid protein sequence is shown in SEQ ID NO:167, which shows the targeting peptide insertion after position 590 as SSAX7AS, where the leading SSA and trailing AS are linker sequences, and X7 represents the targeting peptide. An exemplary modified AAV2 capsid protein sequence is shown in SEQ ID NO:168, which shows the targeting peptide insertion after position 587 as AAAX7AA, where the first AAA and the second AA are linker sequences, and X7 represents the targeting peptide. An exemplary modified AAV9 capsid protein sequence is shown in SEQ ID NO:169, which shows the targeting peptide insertion after position 588 as AAAX7AS, where the first AAA and the second AS are linker sequences, and X7 represents the targeting peptide.

[0035] I. Adeno-associated virus (AAV) vectors Adeno-associated viruses (AAVs) are small, non-pathogenic viruses of the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, with more than a dozen isolated from humans or primates. AAVs differ from other members of the family in that they depend on a helper virus to replicate.

[0036] The AAV genome exists in an extrachromosomal state without integration into the host cell genome, has a broad host range, and can 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 thermostable, resistant to solvents, detergents, pH changes, and temperature, and can be column purified and / or concentrated by CsCl gradients or other means. The AAV genome comprises positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb genome of AAV consists of one segment of single-stranded DNA of positive or negative polarity. At the end of the genome are short inverted terminal repeats (ITRs) that fold into a hairpin structure and serve as origins of viral DNA replication.

[0037] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or enclosed to form an AAV particle. AAV particles often contain the AAV genome packaged by AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not contain the "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is called the "plasmid backbone", which is important for the cloning and amplification of the plasmid, a process required for the growth and production of the plasmid, but is not itself packaged or enclosed in the viral particle. Thus, AAV vector "genome" refers to the nucleic acid that is packaged or enclosed by AAV capsid proteins.

[0038] AAV virions (particles) are non-enveloped icosahedral particles with a diameter of approximately 25 nm that contain the AAV capsid. AAV particles contain icosahedral symmetry composed of three associated capsid proteins VP1, VP2, and VP3, which interact together to form the capsid. The genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as the left and right ORFs. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a 1:1:10 ratio, respectively, but can be found in a variety of ratios, all originating from the right ORF. The VP1, VP2, and VP3 capsid proteins differ from each other in the use of alternative splicing and rare start codons. Deletion analysis has shown that removal or alteration of VP1, which is translated from the alternatively spliced ​​message, reduces the yield of infectious particles. Mutations within the VP3 coding region result in neither single-stranded progeny DNA nor infectious particles. In certain embodiments, the AAV particle genome encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0039] The left ORF often encodes non-structural Rep proteins, Rep40, Rep52, Rep68, and Rep78, which are involved in regulating replication and transcription as well as generating single-stranded progeny genomes. Two of the Rep proteins are associated with preferential integration of AAV genomes into the region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity and DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and several potential phosphorylation sites. In certain embodiments, the genome of an AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of an AAV (e.g., rAAV) does not encode a Rep protein. In certain embodiments, one or more of the Rep proteins can be delivered in trans and thus are not included in the AAV particle that contains the nucleic acid encoding a polypeptide.

[0040] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into a T-shaped hairpin structure that serves as an origin of viral DNA replication. Thus, the AAV genome contains one or more (e.g., a pair) ITR sequences flanking the single-stranded viral DNA genome. The ITR sequences are often about 145 bases each in length. Within the ITR region, two elements that are thought to be central to the function of the ITRs have been described: the GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs in a site-specific and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. The Rep binding site and the adjacent trs are contained within the chromosome 19 integration locus. These elements have been shown to function and be required for locus-specific integration.

[0041] The term "recombinant" as a modifier of vectors, such as recombinant virus, e.g., lentivirus or parvovirus (e.g., AAV) vectors, and as a modifier of sequences, such as recombinant nucleic acid sequences and recombinant polypeptides, means that the composition has been manipulated (i.e., engineered) in a manner that does not normally occur in nature. A particular example of a recombinant vector, such as an AAV, retrovirus, or lentivirus vector, would be a recombinant vector in which a nucleic acid sequence that is not normally present in the wild-type viral genome has been inserted into the viral genome. An example of a recombinant nucleic acid sequence would be a recombinant nucleic acid sequence encoding an inhibitory RNA in which a nucleic acid (e.g., a gene) has been cloned into the vector, with or without the 5', 3', and / or intron regions with which the gene is normally associated with the viral genome. The term "recombinant" is not always used herein with respect to vectors, such as viral vectors, and sequences, such as polynucleotides, although "recombinant" forms, including nucleic acid sequences, polynucleotides, transgenes, and the like, are expressly included despite such omission.

[0042] A recombinant viral "vector" is derived from a wild-type viral genome by using molecular methods to remove a portion of the wild-type genome from the virus and replace it with a non-natural nucleic acid, such as a nucleic acid sequence. Typically, for example, in the case of AAV, one or both inverted terminal repeats (ITRs) 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 because a portion of the viral genome is replaced with a non-natural sequence, such as a nucleic acid encoding a transactivator or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein, relative to the viral genome nucleic acid. Thus, the incorporation of such a non-natural nucleic acid sequence defines the viral vector as a "recombinant" vector, and in the case of AAV, the "recombinant" vector may be referred to as a "rAAV vector."

[0043] In certain embodiments, an AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises at least a pair of ITRs adjacent to (i.e., at the 5' and 3' ends of) a nucleic acid sequence encoding a polypeptide having a function or activity.

[0044] AAV vectors (e.g., rAAV vectors) can be packaged and subsequently infected (transduced) into cells, ex vivo, in vitro, or in vivo, and are referred to herein as "AAV particles." When a recombinant AAV vector is enclosed or packaged in an AAV particle, the particle may also be referred to as an "rAAV particle." In certain embodiments, the AAV particle is an rAAV particle. The rAAV particle often includes an rAAV vector or a portion thereof. The rAAV particle may be one or more rAAV particles (e.g., multiple AAV particles). The rAAV particle typically includes proteins (e.g., capsid proteins) that enclose or package the rAAV vector genome. It is noted that references to rAAV vectors can also be used to refer to rAAV particles.

[0045] Any suitable AAV particle (e.g., rAAV particle) can be used for the methods or uses herein. The rAAV particle and / or the genome contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particle and / or the genome contained therein can be derived from two or more serotypes or strains of AAV. Thus, the rAAV can comprise the proteins and / or nucleic acids, or portions thereof, of any serotype or strain of AAV, and the AAV particle is suitable for infection and / or transduction of mammalian cells. 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.

[0046] In certain embodiments, the plurality of rAAV particles comprises particles of or derived from the same strain or serotype (or subgroup or variant), hi certain embodiments, the plurality of rAAV particles comprises a mixture of two or more different (e.g., different serotypes and / or strains) rAAV particles.

[0047] The term "serotype" as used herein is a distinction used to refer to an AAV having a serologically distinct capsid from other AAV serotypes. Serologic distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV when compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Although it is possible that AAV variants, including capsid variants, are not serologically distinct from a reference AAV or other AAV serotypes, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotypes.

[0048] In certain embodiments, an rAAV vector based on a first serotype genome corresponds to one or more serotypes of the capsid protein that packages the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that comprise the AAV vector genome corresponds to the serotype of the capsid that comprises the rAAV particle.

[0049] In certain embodiments, the rAAV vector genome may be based on a different AAV (e.g., AAV2) serotype genome from one or more serotypes of the AAV capsid proteins that package the vector. For example, the rAAV vector genome may contain AAV2-derived nucleic acid (e.g., ITRs), while at least one or more of the three capsid proteins are derived from a different serotype, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype, or a variant thereof.

[0050] In certain embodiments, an rAAV particle or vector genome thereof related to a reference serotype has a polynucleotide, polypeptide, or subsequence thereof that comprises or consists of a sequence that is 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 thereof of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In certain embodiments, an rAAV particle or its vector genome related to a reference serotype has a capsid or ITR sequence that comprises or consists of 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 the capsid or ITR sequences of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0051] In certain aspects, the methods herein include use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0052] In certain embodiments, the methods herein include the use, administration, or delivery of rAAV2 particles.In certain embodiments, the rAAV2 particles include AAV2 capsids.In certain embodiments, the rAAV2 particles include one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid proteins of natural or wild-type AAV2 particles, such as 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 the corresponding capsid proteins of natural or wild-type AAV2 particles. In certain embodiments, the rAAV2 particles comprise VP1, VP2, and VP3 capsid proteins that are at least 75% identical or more identical to the corresponding capsid proteins of a native or wild-type AAV2 particle, 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. In certain embodiments, the rAAV2 particles are variants of a native or wild-type AAV2 particle. In some aspects, one or more capsid proteins of the AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid protein of a native, or wild-type, AAV2 particle.

[0053] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles comprise one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid proteins of a native or wild-type AAV9 particle, 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. In certain embodiments, the rAAV9 particles comprise VP1, VP2, and VP3 capsid proteins that are at least 75% identical or more identical to the corresponding capsid proteins of a native, or wild-type, AAV9 particle, 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. In certain embodiments, the rAAV9 particles are variants of a native, or wild-type, AAV9 particle. In some aspects, one or more capsid proteins of the AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of a native, or wild-type, AAV9 particle.

[0054] In certain embodiments, one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, 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, are provided that are capable of providing one or more desirable ITR functions (e.g., ability to form a hairpin, allowing for DNA replication; ability to integrate an AAV into a host cell genome). The rAAV particles can contain one or two ITRs (e.g., a pair of ITRs), so long as they retain the required DNA integration; and / or packaging, if desired.

[0055] In certain embodiments, the rAAV2 particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITRs of a native, or wild-type, AAV2 particle, 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, so long as the one or two ITRs (e.g., a pair of ITRs) retain one or more desirable ITR functions (e.g., the ability to form a hairpin to enable DNA replication; integration of AAV DNA into the host cell genome; and / or packaging, if desired).

[0056] In certain embodiments, the rAAV9 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITRs of a native, or wild-type, AAV2 particle, 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, so long as the one or two ITRs (e.g., a pair of ITRs) retain one or more desirable ITR functions (e.g., the ability to form a hairpin to enable DNA replication; integration of AAV DNA into the host cell genome; and / or packaging, if desired).

[0057] rAAV particles may include an ITR with any suitable number of "GAGC" repeats. In certain embodiments, the ITR of an AAV2 particle includes one, two, three, four, five, six, seven, eight, nine, or ten or more "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with three "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with fewer than four "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with more than four "GAGC" repeats. In certain embodiments, the ITR of an rAAV2 particle includes a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0058] Exemplary suitable lengths of DNA that can be incorporated into an rAAV vector for packaging / encapsidation into an rAAV particle can be about 5 kilobases (kb) or less, in certain embodiments, the length of the DNA is less than about 5 kb, 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.

[0059] rAAV vectors containing nucleic acid sequences that induce RNAi or polypeptide expression can be produced using suitable recombinant methods known in the art (see, for example, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transduction-competent AAV particles and propagated using AAV viral packaging systems. Transduction-competent AAV particles can bind and enter mammalian cells and then deliver nucleic acid cargo (e.g., heterologous genes) to the cell nucleus. Thus, intact transduction-competent rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often replication-incompetent and require additional protein machinery to replicate themselves. Thus, rAAV particles configured to transduce mammalian cells are engineered to bind and enter mammalian cells and deliver nucleic acid to cells, and the nucleic acid for delivery is often located between a pair of AAV ITRs in the rAAV genome.

[0060] 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 heterologous rAAV vectors. HEK293, a cell derived from a suitable human cell line (e.g., can be readily obtained through the American Type Culture Collection under accession number ATCC CRL1573), can be used. In certain embodiments, modified human embryonic kidney cell lines (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing adenovirus E1a and E1b genes are used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected, providing a particularly convenient platform for producing rAAV particles. Methods for producing high-titer AAV particles that can transduce mammalian cells are known in the art. For example, AAV particles can be made as shown in Wright, 2008 and Wright, 2009.

[0061] In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct prior to or simultaneously with transfection of the AAV expression vector. Thus, AAV helper constructs are sometimes used to transiently express at least the AAV rep and / or cap genes and to complement missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and do not replicate or package themselves. These constructs may take 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 code for both Rep and Cap expression products. A number of other vectors are known that code for Rep and / or Cap expression products.

[0062] An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence together with the necessary regulatory regions required 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 heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0063] II. Therapeutic Agents In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNAs, non-coding RNAs, and / or therapeutic proteins to cells in culture or in a host organism.

[0064] A. Inhibitory RNA "RNA interference (RNAi)" is the process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA and the resulting sequence-specific inhibition of gene expression.

[0065] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecule, "short hairpin RNA" or "shRNA" molecule, or "miRNA" is an RNA duplex of nucleotides that targets a nucleic acid sequence of interest. The term "siRNA" as used herein is a generic term that includes a subset of shRNA and miRNA. "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. The nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene, so that the siRNA is "targeted" to the gene. In certain embodiments, the siRNA is targeted to the sequence that codes for huntingtin. In some embodiments, the length of the duplex of the siRNA is less than 30 base pairs. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs long. In some embodiments, the length of the duplex is 19-25 base pairs long. In certain embodiments, the length of the duplex is 19 or 21 base pairs long. The RNA duplex portion of the siRNA may be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion disposed between the two sequences forming the duplex. The loop may vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In certain embodiments, the loop is 18 nucleotides long. The hairpin structure may also contain a 3' and / or 5' overhang portion. In some embodiments, the overhang is a 3' and / or 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0066] shRNAs are composed of a stem-loop structure designed to contain a 5' flanking region, an siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies have utilized short hairpin RNAs (shRNAs) driven by strong polIII-based promoters. Many shRNAs have demonstrated effective knockdown of target sequences in vitro as well as in vivo. However, some shRNAs that demonstrated effective knockdown of target genes were also found to be toxic in vivo.

[0067] miRNA is a small cellular RNA (about 22nt) that is processed from precursor stem-loop transcript.Known miRNA stem-loop can be modified to contain RNAi sequence specific to gene of interest.Because miRNA is endogenously expressed, miRNA molecule may be preferred over shRNA molecule.Therefore, miRNA molecule is less likely to induce dsRNA-responsive interferon pathway, is more efficiently processed than shRNA, and has been shown to effectively silence 80% more.

[0068] Recently discovered alternative approach is the use of artificial miRNA (pri-miRNA scaffold shuttling siRNA sequence) as RNAi vector.Artificial miRNA is more similar to natural endogenous RNAi substrate, and is amenable to Pol-II transcription (e.g., allows tissue-specific expression of RNAi) and polycistronic strategy (e.g., allows delivery of multiple siRNA sequences).See U.S. Patent No. 10,093,927, which is incorporated by reference.

[0069] The "shRNA" transcription unit consists of sense and antisense sequences connected by a loop of unpaired nucleotides. shRNAs are transported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. "miRNA" stem-loops consist of sense and antisense sequences connected by a loop of unpaired nucleotides that are typically expressed as part of a larger primary transcript (pri-miRNA) and are excised by the Drosha-DGCR8 complex to generate an intermediate known as the pre-miRNA. This intermediate is then transported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. As used interchangeably herein, "artificial miRNA" or "artificial miRNA shuttle vector" refers to a primary miRNA transcript in which the region of the double-stranded stem-loop (at least about 9-20 nucleotides) excised by Drosha and Dicer processing has been replaced with an siRNA sequence for a target gene, while retaining the structural elements within the stem-loop required for effective Drosha processing. The term "artificial" comes from the fact that the flanking sequences (about 35 nucleotides upstream and about 40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences as well as artificially created miRNA shuttle vectors.

[0070] siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.The nucleic acid sequence can also include a polyadenylation signal.In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six T's.

[0071] When designing RNAi, there are several factors that need to be taken into account, such as the nature of siRNA, the durability of silencing effect, and the choice of delivery system. To produce RNAi effect, the siRNA that is introduced into an organism typically contains exon sequence. Furthermore, the RNAi process depends on homology, so the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA shows greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the sequence of the siRNA and the sequence of the gene to be inhibited. Sequences with less than about 80% identity to the target gene are substantially ineffective. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0072] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19-25 nucleotides and can regulate gene expression. In the context of the present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0073] siRNA target generally refers to a polynucleotide that comprises a region that codes for a polypeptide, or a polynucleotide region that regulates the replication, transcription, or translation or other processes that are important for the expression of the polypeptide, or a polynucleotide that comprises both a region that codes for a polypeptide and a region that regulates expression that is functionally linked to the region that codes for the polypeptide.Any gene that is expressed in cells can be targeted.Preferably, the target gene is a gene that is involved in or related to the progression of a cellular activity that is important for disease or that is of particular interest for research.

[0074] B. Non-coding RNA More than 90% of the human genome is transcribed but does not code for proteins, as evidenced by cDNA cloning projects and genome tiling arrays. These transcripts are called non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNAs, transfer RNAs, competing endogenous RNAs (ceRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs), are essential for cellular functions. Similarly, a number of short ncRNAs, such as microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that exhibit cell type-specific expression and are localized to specific subcellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the idea that they have been selected during the evolutionary process.

[0075] lncRNAs appear to have many different functions. In many cases, they appear to play a role in regulating protein activity or localization, or serve as an organizational framework for subcellular structures. In other cases, lncRNAs may be processed to produce multiple small RNAs, or they may regulate the way other RNAs are processed. The latest data compiled by the public research consortium GenCode (version #27) lists just under 16,000 lncRNAs in the human genome that produce nearly 28,000 transcripts. Including other databases, more than 40,000 lncRNAs are known.

[0076] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, modulate the activity of protein-binding partners, target chromatin-modifying complexes to their sites of action, and are post-transcriptionally processed to generate numerous 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0077] A growing body of evidence also suggests that aberrantly expressed lncRNAs play important roles in normal physiological processes as well as multiple disease states. lncRNAs are misregulated in various diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation has also been shown in various types of cancer, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Some lncRNAs, e.g., gadd74 and lncRNA-RoR5, regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thus providing an additional layer of versatility and robustness to cell cycle progression. In addition, some lncRNAs have been associated with mitotic processes, such as centromeric satellite RNAs, which are essential for kinetochore formation in humans and flies and thus important for chromosome segregation during mitosis. Another nuclear lncRNA, MA-lincl, regulates exit from M phase by functioning in cis to repress the expression of its neighboring gene, Pura, a cell proliferation regulator.

[0078] lncRNAs are a group generally defined as transcripts larger than 200 nucleotides (e.g., about 200 to about 1200 nt, about 2500 nt, or more) that lack an extended open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes lncRNAs as well as shorter transcripts, e.g., less than about 200 nt, e.g., about 30 to 200 nt.

[0079] Thus, in some embodiments, delivery of ncRNA, for example, to a particular brain structure of interest, corrects ectopic RNA expression levels or modulates disease-causing lncRNA levels. Thus, in some embodiments, the present invention provides rAAV, where the viral genome is engineered to encode a therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) that is about 200 nucleotides (nt) long or longer. In some embodiments, the therapeutic agent is an ncRNA that is about 25 nt or about 30 nt to about 200 nt long. In some embodiments, the lncRNA is about 200 nt to about 1,200 nt long. In some embodiments, the lncRNA is about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nt long.

[0080] C. CRISPR Systems Gene editing is a technique that allows targeted genetic modification within living cells. Recently, harnessing the CRISPR bacterial immune system to perform gene editing on demand has revolutionized the way scientists approach genome editing. The Cas9 protein in the CRISPR system is an RNA-guided DNA endonuclease that can be relatively easily engineered to target new sites by altering its guide RNA sequence. This discovery has functionally enabled sequence-specific gene editing.

[0081] Generally, a "CRISPR system" refers collectively to the transcripts and other elements involved in the expression of or that induce the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr-mate sequences (which, in the context of an endogenous CRISPR system, encompass "direct repeats" and partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0082] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system derived from a particular organism that contains an endogenous CRISPR system, such as, for example, Streptococcus pyogenes.

[0083] CRISPR systems can induce and subsequently destroy double-strand breaks (DSBs) at target sites, as discussed herein. In other embodiments, Cas9 variants considered "nickases" are used to nick one strand at the target site. For example, to improve specificity, paired nickases can be used, each nickase being directed by a pair of different gRNA targeting sequences such that a 5' overhang is introduced when the nicks are introduced simultaneously. 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, the CRISPR system comprising catalytically inactive Cas9 further comprises a transcriptional repressor or activator fused to a ribosome-binding protein.

[0084] In some aspects, Cas nuclease and gRNA (comprising a fusion of a crRNA specific for a target sequence and a certain tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site may be selected based on being immediately 5' to a protospacer adjacent motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, the gRNA is targeted to a 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, CRISPR systems feature elements that promote the formation of a CRISPR complex at the target sequence site. Typically, a "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization of the target sequence with the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not required, provided there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

[0085] The target sequence may comprise any polynucleotide, for example, a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, for example, in an organelle of a cell. In general, the sequence or template that can be used for recombination into a target locus that comprises a target sequence is called an "editing template" or an "editing polynucleotide" or an "editing sequence". In some aspects, the exogenous template polynucleotide may be called an editing template. In some aspects, the recombination is a homologous recombination.

[0086] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (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 may also comprise or consist of all or a portion of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence, or about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides longer than the wild-type tracr sequence), and may form part of a CRISPR complex, for example, by hybridizing to all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0087] One or more vectors that drive the expression of one or more elements of the CRISPR system can be introduced into the cell, so that the expression of the elements of the CRISPR system induces the formation of a CRISPR complex at one or more target sites. Components can also be delivered to the cell as protein and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. The Cas enzyme can be a target gene under the control of regulated alternative splicing events as disclosed herein, as a chimeric target gene minigene, or as a target gene of a chimeric minigene transactivator. The gRNA can be under the control of a constitutive promoter.

[0088] Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in one vector, and one or more additional vectors may provide any components of the CRISPR system that are not included in the first vector. The vector may include one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). 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 in a cell.

[0089] The vector may include a regulatory element operably linked to a CRISPR enzyme, e.g., an enzyme coding sequence encoding 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 amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0090] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme may, for example, induce cleavage of one or both strands within the target sequence and / or the complementary strand of the target sequence at the location of the target sequence. The vector may encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid 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 (that cleaves a single strand). In some embodiments, the Cas9 nickase may be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target. This combination allows for the creation of nicks on both strands and can be used to induce NHEJ or HDR.

[0091] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be a eukaryotic cell of a particular organism, such as a mammalian eukaryotic cell, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate, or may be derived from such a eukaryotic cell. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a more frequently or most frequently used codon in the host cell's genes, while maintaining the native amino acid sequence, for enhanced expression in the host cell of interest. Different species show a certain bias for a certain codon of a certain amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, and as a result, it is believed that the efficiency of messenger RNA (mRNA) translation depends, among other things, on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The predominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis, and thus genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0092] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of CRISPR complex to the target sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or is greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more.

[0093] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available from soap.genomics.org.cn), and Maq (available from maq.sourceforge.net).

[0094] CRISPR enzymes may be part of fusion proteins that contain one or more heterologous protein domains. CRISPR enzyme fusion proteins may contain any additional protein sequences, and optionally linker sequences between any two domains. Examples of protein domains that can be fused with CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have 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) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes may be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or bind to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that may form part of fusion proteins containing CRISPR enzymes are described in US20110059502, which is incorporated herein by reference.

[0095] D. Therapeutic Proteins Some embodiments relate to the expression of recombinant proteins and polypeptides. Such proteins may be otoprotective proteins, such as anti-apoptotic proteins, antioxidant enzymes (e.g., those belonging to the superoxide dismutase (SOD) family), neurotrophic / neuroprotective factors, anti-inflammatory proteins, or proteins that promote hair cell regeneration in the vestibular system. The therapeutic proteins include Birc1a (NAIP), Birc2 (c-IAP1 / HIAP-2), Birc3 (cIAP-2 / HIAP-1), Birc4 (XIAP), Birc5 (survivin), Birc6 (apollon), Birc7 (livin), and Birc8 (TsIAP); members of the Bcl-2 family: Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bcl-2L10, and BFL-1; and endogenous c-Jun N-terminal kinase (JNK) kinases, also known as Jun-interacting proteins (JIPs), JIP-1, JIP-2, JIP-3, and JIP-4. inhibitors; 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), NF-κB inhibitor (IκB); USH1, USH1G (also known as 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, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14 , CLIC5, CLPP, CLRNl, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIA PH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, G RXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2 , MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MY015A, MYOIA, MY03A, MY06, MY07A, NARS2, NDP, NF2, NT3, OSBPL 2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SE The therapeutic protein may be MA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGRl, WFS1, WHRN, or XIAP. Additional exemplary therapeutic proteins and associated conditions are listed in Table A.

[0096] [Table A] C: Cochlear-hearing impairment V: Vestibular system disorder

[0097] Further conditions and associated genetic defects are described in the art, see, e.g., Kemperman et al., JR 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.

[0098] Thus, when the present application refers to a function or activity of a "modified protein" or "modified polypeptide," it will be understood by one of skill in the art to include, for example, proteins or polypeptides that have additional advantages over an unmodified protein or polypeptide. It is specifically intended that embodiments relating to a "modified protein" may be implemented with respect to a "modified polypeptide," and vice versa.

[0099] Recombinant proteins may have deletions and / or substitutions of amino acids. Thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further include proteins with inserted or added amino acids, e.g., fusion proteins or linkers. A "modified deletion protein" may lack one or more residues of the native protein, but may have the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. One example of a modified deletion protein is one in which amino acid residues have been deleted from at least one antigenic region, i.e., a region of the protein that has been determined to be antigenic in a particular organism, e.g., the organism to which the modified protein is administered.

[0100] Substitution or exchange variants typically contain an 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. The substitutions may be conservative, i.e., an amino acid is replaced with an amino acid of similar shape or charge, but may not be conservative. Conservative substitutions are well known in the art and include, for example, exchanges of alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; 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.

[0101] In addition to deletion or substitution, modified proteins may have residue insertions, which typically involve the addition of at least one residue to the polypeptide. This may include the insertion of a targeting peptide or polypeptide, or may simply include the insertion of a single residue. Terminal additions, called fusion proteins, are discussed below.

[0102] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, it includes sequences having about 70% to about 80% of the amino acids identical to or functionally equivalent to the amino acids of a reference polypeptide, or about 81% to about 90% of the amino acids, or even about 91% to about 99% of the amino acids, provided that the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be biologically functionally equivalent to its native counterpart.

[0103] It is also understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, but still be essentially as shown in one of the sequences disclosed herein, so long as they meet the criteria set forth above, including the maintenance of biological protein activity related to protein expression. The addition of terminal sequences applies in particular to nucleic acid sequences that may contain, for example, various non-coding sequences adjacent to the 5' or 3' portion of the coding region, or may contain various internal sequences, i.e., introns, which are known to be internal to genes.

[0104] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0105] As used herein, "amino acid residue" refers to any natural amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous and do not interrupt the amino acid residue sequence with non-amino acids. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0106] Thus, the term "protein or peptide" includes an amino acid sequence that includes at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0107] Certain aspects of the present invention relate to fusion proteins. These molecules may have the N-terminus or C-terminus of a therapeutic protein linked to a heterologous domain. For example, fusions may also use leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Another useful fusion 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 a cleavable antibody epitope to facilitate fusion protein purification. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0108] Methods for making fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of a complete fusion protein or by attaching a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0109] The production of a fusion protein that restores the functional activity of the parent protein may be facilitated by joining the genes with a bridging DNA segment that encodes a peptide linker that is spliced ​​between the tandemly connected polypeptides, the linker being of sufficient length to allow for correct folding of the resulting fusion protein.

[0110] The expression of a transgene may be induced by the native promoter of the transgene (i.e., the promoter found in nature with the transgenic coding sequence), or the expression of a transgene may be induced by a heterologous promoter (e.g., CMV promoter, Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter). For example, any transgene described herein may be used with its native promoter. Alternatively, any transgene described herein may be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally induce the expression of the sequence (i.e., is not found in nature with the sequence). Representative heterologous promoters that can be used to induce the expression of any transgene described herein include, for example, CMV promoter, CBA promoter, CASI promoter, P promoter, and EF-1 promoter, alpha9 nicotinic receptor promoter, prestin promoter, Gfil promoter, and Vglut3 promoter. In addition, a promoter that naturally induces the expression of one of the above transgenes (e.g., KCNQ4 promoter, Myo7a promoter, Myo6 promoter, or Atohl promoter) can be used as a heterologous promoter to induce the expression of the transgene. In other embodiments, the promoter is Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter.

[0111] III. Methods of Treatment and Administration Viral vectors may in some aspects be administered directly to a patient (in vivo) or can be used to treat cells in vitro or ex vivo and then administered to a patient. In particular, methods are provided herein for inducing expression of a transgene in cells of the inner ear. In some embodiments, the cells are hair cells of the cochlea or vestibular system. In some embodiments, the cells are inner hair cells of the cochlea or outer hair cells of the cochlea. In some of these embodiments, the subject has hearing loss and the transgene is delivered in a therapeutically effective amount. In some embodiments, the AAV vector transduces at least about 70% of the cells of the inner ear. The AAV vector targets inner and outer hair cells with at least about 70%, 80%, 90%, 95% or more efficiency, and even greater than 100% efficiency. In some embodiments, the cells are cells of the vestibular system, such as hair cells of the utricle, or cells in the ampulla of the lateral semicircular canal, or hair cells at the apex. In some aspects, 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.

[0112] There are two types of hair cells in the cochlea. Inner hair cells (IHCs) convert mechanical stimuli of sound vibrations into neural signals transmitted to the brain by type I spiral ganglion neurons. Outer hair cells (OHCs) are connected only to poorly characterized type II neurons. Their main function is to amplify sound-induced vibrations in a frequency-specific manner by as much as 60 decibels (dB), which is essential for frequency discrimination (important in speech recognition). Most hearing loss genes known to affect hair cell function are expressed in both cell types, so a useful gene therapy strategy must generally target both IHCs and OHCs. Cells of the vestibular system, including hair cells in the semicircular canals (horizontal, anterior, and posterior) or the two otolithic organs (saccule and utricle), are essential for our sense of balance and for coordinating eye movements. These cells are often affected in hereditary hearing loss, so gene therapy must also target them.

[0113] The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by the insertion or incorporation of a nucleic acid. A vector, such as a viral vector, can be used to introduce / transfer a nucleic acid sequence into a cell so that the nucleic acid sequence therein is transcribed and, if the nucleic acid sequence encodes a protein, then translated by the cell.

[0114] These compositions can be used to treat conditions associated with hearing loss or vestibular dysfunction, which conditions are caused by genetic defects or ameliorated by gene therapy. Thus, in some embodiments, the methods described herein are used to treat the conditions listed in Table A in a subject in need of treatment, using the corresponding sequence listing in Table A. Examples include certain types of Usher syndrome (blindness and hearing loss associated with some types of vestibular dysfunction).

[0115] In one example, the hearing loss is presbycusis. In another example, the hearing loss is high frequency hearing loss. High frequency hearing loss occurs above 2 kHz. In yet another example, the hearing loss is due to ototoxicity, noise-induced hearing loss, viral infection of the inner ear, autoimmune inner ear disease, genetic hearing loss, inner ear barotrauma; physical or surgical trauma; or inflammation. The ototoxicity is due to cisplatin treatment of a subject suffering from cancer. In one example, the genetic hearing loss is Usher I syndrome, Usher II syndrome, or Usher III syndrome. In one example, the balance disorder occurs in an elderly subject. In another example, the vestibular dysfunction is the result of degeneration of the vestibular apparatus. The regeneration of the vestibular apparatus is due to ototoxicity, viral infection of the inner ear, autoimmune inner ear disease, genetic vestibular loss, inner ear barotrauma; or physical or surgical trauma.

[0116] For example, genetically based hearing loss is a significant problem with few treatment options other than cochlear implants. Hereditary hearing problems are often due to a single gene defect. Prelingual hearing loss is diagnosed in 1 / 500 infants, of which approximately 50% have a genetic etiology. Usher syndrome is associated with a number of different clinical subtypes, each of which can be caused by mutations in any of a number of different genes, and is responsible for 3-6% of early childhood hearing loss. One of the predominant gene defects, estimated to account for 1-2% of all genetic hearing loss, occurs in the TMC1 gene. Usher syndrome is classified into three clinical subtypes (USH-1, -2, and -3) according to the severity of the symptoms. USH1 is the most severe form. Patients affected by USH1 suffer from congenital bilateral profound sensorineural hearing loss, vestibular areflexia, and prepubertal retinitis pigmentosa (progressive, bilateral, symmetric degeneration of retinal rod and cone function). Individuals typically do not develop language skills unless a cochlear implant is installed. Currently, there is no biological treatment for Usher patients, but early reintroduction of the wild type of the defective gene may allow for reversal of the disease.

[0117] USH1, the most severe form of Usher syndrome, 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 protein 2). These genes code for proteins that are involved in hair bundle morphogenesis in the inner ear and are part of the interactome (see, for example, Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20). Harmonin is central to the USH1 interactome and binds to other Usher1 proteins. Due to its PDZ (PSD-5995 / Dlg / ZO-l) interacting domain, harmonin has been proposed to function as a scaffolding protein. In vitro binding studies have shown that all other known USH1 proteins bind to the PDZ domain of harmonin, as do two of the USH2 proteins, usherin and VLGRl. The USHI C gene consists of 28 exons and encodes 10 alternatively spliced ​​forms of harmonin, grouped into three distinct 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-coil (CC) domains, and proline-serine-threonine (PST)-rich domains. The USH1 protein is localized at the apical end of hair cells in mechanosensory hair bundles, which are composed of hundreds of stereocilia interconnected by numerous extracellular tethers. Cadherin 23 and protocadherin 15 are products of the Usher gene (USHI D and USH1 E, respectively) and form the tip tethers located at the distal end of the stereocilia. Harmonin-b binds to CDH23, PCDH15, F-actin, and itself. Harmonin-b is found at the tips of stereocilia near the insertion points of the tip junctions in hair cells, where it is thought to play a functional role in transmission and adaptation in hair cells.Harmonin-b is expressed during early postnatal stages, but its expression declines at approximately postnatal day 30 (P30) in both the cochlea and vestibule. Harmonin-a also binds cadherin 23 and is found in stereocilia. A recent report uncovered an additional role for harmonin-a at synapses, where it binds to Cavl.3 Ca2+ channels and restricts their availability via a ubiquitin-dependent pathway.

[0118] Several mouse models of Usher syndrome have been identified or engineered over the past decade, seven of which affect harmonin. Of these, only one model, the Ushlc c.216G>A model, recapitulates both the hearing and retinal defects that characterize human Usher syndrome. Ushlc c.216G>A is a knock-in mouse model that affects the expression of all conventional harmonin isoforms due to a point mutation similar to that found in the French-Acadian USHI C patient cohort. This mutation introduces a cryptic splice site at the end of exon 3 of the Ushlc gene. Usage of this cryptic splice site results in a frameshifted transcript with a 35bp deletion, leading to the translation of a severely truncated protein lacking the PDZ, PST, and CC domains. Homozygous C.216AA knock-in mice suffer from profound hearing loss at 1 month of age, whereas heterozygous C.216GA mice do not display any abnormal phenotype. C.21 Cochlear histology in AA mice shows disorganized hair bundles, abnormal cell rows, and loss of inner and outer hair cells in the middle and basal turns at P30.

[0119] More than 40 different mutations have been identified in TMC1 that cause hearing loss. These are further divided into 35 recessive and 5 dominant mutations. Most recessive mutations cause severe congenital hearing loss (e.g., DFNB7 / 11), but some cause late-onset moderate to severe hearing loss. All dominant mutations cause progressive hearing loss with onset in the mid-teens (e.g., DFNA36). In particular, AAV vectors containing the Anc80 capsid protein described herein can be used to deliver non-mutated (e.g., wild-type) TMC1 or TMC2 sequences, thereby preventing hearing loss (e.g., further hearing loss) and / or restoring hearing function.

[0120] Therapeutic gene transfer into the cochlea has been considered as an improvement over the current standard of care, ranging from age-related and environmentally induced hearing loss to genetic hearing loss. Over 300 genetic loci have been associated with hereditary hearing loss, and over 70 causative genes have been described (Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). Therapeutic success in these approaches relies heavily on the safe and efficient delivery of exogenous gene constructs to the relevant therapeutic cellular targets in the organ of Corti (OC) of the cochlea. The OC contains two types of sensory hair cells: IHCs, which convert mechanical information carried by sound into electrical signals transmitted to neuronal structures, and OHCs, which help amplify and modulate the cochlear response, a process necessary for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells at the edge of the spiral lamina that are important for maintaining the adjacent tectorial membrane, or supporting cells that have a protective function and can be induced to transdifferentiate into hair cells up to the early neonatal stage.

[0121] As used herein, inner ear cells refer to, but are not limited to, 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. One example of a supporting cell is a Schwann cell.

[0122] Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein is suspected of having or expressing an abnormal or ectopic protein associated with a disease state. Alternatively, the mammalian recipient may have a condition amenable to gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration to the recipient of exogenous genetic material encoding a therapeutic agent in situ, followed by expression of the administered genetic material. Thus, the phrase "condition amenable to gene replacement therapy" encompasses conditions such as genetic diseases (i.e., disease conditions resulting from one or more gene defects) and acquired pathologies (i.e., pathological conditions not resulting from congenital defects). Thus, as used herein, the term "therapeutic agent" refers to any drug or material that has a beneficial effect on the mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules having a nucleic acid or protein component.

[0123] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal may be of any age and at any stage of development (e.g., an adult, teenager, child, infant, or in utero mammal). The mammal may be male or female. In certain aspects, the mammal may be an animal disease model, e.g., an animal model having or expressing an abnormal or ectopic protein associated with a disease state, or an animal model having insufficient protein expression that causes a disease state.

[0124] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. A typical adult is 50 years of age or older. Children are 1-2 years of age, or 2-4 years of age, 4-6 years of age, 6-18 years of age, 8-10 years of age, 10-12 years of age, 12-15 years of age, and 15-18 years of age. Children also include infants. Infants are typically 1-12 months of age.

[0125] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, and the severity, frequency, progression, or onset time of one or more symptoms of a disease state, such as a neurodegenerative disease, is reduced, reduced, prevented, inhibited, or delayed.In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat a deleterious symptom of a disease state, such as a neurodegenerative disease.In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to stabilize, delay, or prevent the deterioration or progression of a disease state, such as a neurodegenerative disease, or to reverse a deleterious symptom.

[0126] In certain aspects, the methods include administering a plurality of viral particles, as provided herein, to the central nervous system of a mammal, or a portion thereof, and the severity, frequency, progression, or time to onset of one or more symptoms of a disease state, such as a neurodegenerative disease, is reduced, reduced, prevented, inhibited, or delayed by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days.

[0127] In some embodiments, a composition can be delivered that includes a therapeutically effective number of viral particles that contain a transgene or that contain one or more sets of different viral particles, where each particle in a set may contain the same type of transgene, but each set of particles contains a different type of transgene than the other sets, as described herein.

[0128] Formulations according to the present invention can be used for CNS delivery via a variety of techniques and routes, including, but not limited to, intraparenchymal, intracerebral, intravetricular cerebral (ICV), intrathecal (e.g., IT-lumbar, IT-thoracic, IT-cisternal) administration, and any other technique and route for direct or indirect injection into the CNS and / or CSF.

[0129] In some embodiments, the formulation is delivered to the CNS by administration to the cerebrospinal fluid (CSF) of the subject in need of treatment. In some embodiments, intrathecal delivery is used to deliver viral particles to the CSF. As used herein, intrathecal administration (also called intrathecal injection) refers to injection into the spinal canal (the intrathecal space surrounding the spinal cord). Various techniques can be used, including but not limited to lateral ventricle injection through a cranial burr hole or cisternal puncture or lumbar puncture. 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.

[0130] According to the present invention, the viral particles can be injected into any area surrounding the spinal canal. In some embodiments, the viral particles are injected into the lumbar region or cisterna magna, or intraventricularly into the ventricular space. As used herein, the term "lumbar region" or "lumbar site" refers to the site between the third and fourth lumbar vertebrae (low back), more generally the L2-S1 region of the spine. Typically, intrathecal injection via the lumbar region or lumber site is also referred to as "lumbar IT delivery" or "lumbar IT administration". The term "cistern magna" refers to the space surrounding and below the cerebellum, through the opening between the skull and the top of the spine. Typically, intrathecal injection via the cisterna magna is also referred to as "cisternal delivery". The term "ventricle" refers to the cavity in the brain that is continuous with the central canal of the spinal cord. Thus, intrathecal delivery includes any injection into the central canal. Typically, injection through the ventricular cavity is called intracerebroventricular (ICV) delivery.

[0131] Various devices can be used for intrathecal delivery according to the present invention. In some embodiments, the device for intrathecal administration comprises a fluid access port (e.g., injection 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 the spinal cord; and a fixing mechanism for fixing the insertion of the hollow body into 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., infusion pump). In some embodiments, an implanted catheter is connected to either the reservoir (e.g., for bolus delivery) or the infusion pump. The fluid access port can be implanted or external.

[0132] In some embodiments, intrathecal administration may be by lumbar puncture (i.e., slow bolus) or via a port-catheter delivery system (i.e., infusion or bolus). In some embodiments, a catheter is inserted between the lumbar laminae and the tip is threaded up the thecal space to the desired level (typically L3-L4).

[0133] A single dose volume suitable for intrathecal administration is typically small. Typically, intrathecal delivery according to the present invention maintains equilibrium of the composition of the CSF as well as the intracranial pressure of the subject. In some embodiments, intrathecal delivery is performed without a corresponding removal of CSF from the subject. In some embodiments, a suitable single dose volume may be, for example, 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 first involves removing a desired amount of CSF. 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 initially removed prior to IT administration. In these cases, an appropriate single dose volume may be, for example, greater than about 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

[0134] Various other devices can be used to effect intrathecal administration of therapeutic compositions. For example, a formulation containing a desired enzyme can be administered using an Ommaya reservoir, which is commonly used to intrathecally administer 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 connected to an Ommaya reservoir placed under the scalp, which is subcutaneously punctured to intrathecally deliver the specific enzyme that is infused into the reservoir and exchanged. Other devices for intrathecally delivering therapeutic compositions or formulations to individuals are described in U.S. Patent No. 6,217,552, which is incorporated herein by reference. Alternatively, viral particles can be administered intrathecally, for example, by a single injection or continuous infusion. It should be understood that the administration can take the form of a single dose administration or a multiple dose administration.

[0135] In one embodiment of the present invention, viral particles are administered by injection into the lateral ventricle of the subject's brain. Injection can be, for example, through a cranial burr hole made in the subject's skull. In another embodiment, viral particles and / or other pharmaceutical preparations are administered into the subject's ventricle through a surgically inserted shunt. For example, injection can be into the larger lateral ventricle. In some embodiments, injection can also be into the smaller third and fourth ventricles. In yet another embodiment, the pharmaceutical composition used in the present invention is administered by injection into the cisterna magna or into the lumbar region of the subject.

[0136] IV. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable composition, formulation, liquid, or solid, or mixture thereof, 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 undesirable or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions may be sterile. Such pharmaceutical formulations and compositions may be used, for example, in administering viral particles to a subject.

[0137] 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 agents, and absorption enhancers or delayers that are 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 agents, antiviral agents, and antifungal agents) can also be incorporated into the formulations and compositions.

[0138] Pharmaceutical compositions typically contain pharmaceutically acceptable excipients. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts, such as salts of mineral acids, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like, may be included therein. In addition, auxiliary substances, such as surfactants, wetting agents or emulsifiers, pH buffering substances, and the like, may be present in such vehicles.

[0139] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0140] Pharmaceutical forms suitable for injection or infusion of viral particles may include sterile aqueous solutions or dispersions, optionally encapsulated in liposomes, adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final form must be sterile liquid and stable under the conditions of manufacture, use, and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using surfactants. Isotonic agents, for example, sugars, buffers, or salts (for example, sodium chloride), can be included. Prolonged absorption of the injectable composition can be achieved by using absorption-delaying agents, for example, aluminum monostearate and gelatin, in the composition.

[0141] The solution or suspension of viral particles may optionally contain one or more of the following components: a sterile diluent, e.g., water for injection, saline, e.g., phosphate buffered saline (PBS), artificial CSF, surfactants, fixed oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents; antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates, and agents for adjusting tonicity, e.g., sodium chloride or dextrose.

[0142] Pharmaceutical formulations, compositions, and delivery systems suitable for use in the compositions, methods, and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20).th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0143] Viral particles and compositions thereof may be formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to a physically separate unit suitable as a unitary dosage for an individual to be treated. Each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a required pharmaceutical carrier. The unit dosage form depends on the number of viral particles that are considered to be required to produce a desired effect. The required amount may be formulated in a single dose or in multiple dosage units. The dose may be adjusted to a suitable viral particle concentration, optionally in combination with an anti-inflammatory agent, and packaged for use.

[0144] In one embodiment, the pharmaceutical composition contains sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate the symptoms or side effects of the disease state in question, or an amount sufficient to impart a desired benefit.

[0145] As used herein, "unit dosage form" refers to a physically separate unit suitable for the subject to be treated as a unitary dosage. Each unit contains a predetermined amount calculated to produce a desired effect (e.g., preventive or therapeutic effect) when administered in one or more doses, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle, or filler). The unit dosage form may be, for example, in ampoules and vials, which may contain a liquid composition or may contain the composition in a freeze-dried or lyophilized state. For example, a sterile liquid carrier may be added before administration or delivery in vivo. Individual unit dosage forms may be placed in a multi-dose kit or container. Thus, for example, the virus particles and pharmaceutical compositions thereof may be packaged in one or more unit dosage forms for ease of administration and uniformity of dosage.

[0146] A formulation containing viral particles typically contains an effective amount, which is easily determined by one skilled in the art. The viral particles typically comprise about 1% to about 95% (w / w) of the composition, or even more if appropriate. The amount to be administered depends on factors such as the age, weight, and physical condition of the mammalian or human subject for which treatment is being considered. An effective dosage can be determined by one skilled in the art by routine testing to determine dose-response curves.

[0147] V. Definition The terms "polynucleotide", "nucleic acid", and "transgene" are used interchangeably herein to refer to all types of nucleic acids, oligonucleotides including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and polymers thereof. Polynucleotides include genomic DNA, cDNA, and antisense DNA, as well as 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 natural, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded, or triplexed, linear or circular, and of any suitable length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of showing the sequence in the 5' to 3' direction.

[0148] A nucleic acid that encodes a polypeptide often includes an open reading frame that encodes the polypeptide. Unless otherwise specified, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0149] The nucleic acid may include one or more expression control or regulatory elements operably linked to the open reading frame, the one or more regulatory elements configured to direct 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, TATA boxes, etc.), translation initiation sequences, mRNA stability sequences, polyA sequences, secretion sequences, etc. Expression control / regulatory elements can be obtained from the genome of any suitable organism.

[0150] "Promoter" refers to a nucleotide sequence, usually upstream (5') of a coding sequence, that induces and / or controls the expression of the coding sequence by recognizing RNA polymerase and other factors necessary for correct transcription. PolII promoters contain a minimal promoter, which is a short DNA sequence consisting of a TATA box and, optionally, other sequences that serve to specify the transcription start point, to which regulatory elements are added for expression control. Type 1 polIII promoters contain three cis-acting sequence elements downstream of the transcription start point: a) 5' sequence element (A block); b) intermediate sequence element (I block); c) 3' sequence element (C block). Type 2 polIII promoters contain two essential cis-acting sequence elements downstream of the transcription start point: a) A box (5' sequence element); and b) B box (3' sequence element). Type 3 polIII promoters contain several cis-acting promoter elements upstream of the transcription start point, such as a conventional TATA box, a proximal sequence element (PSE), and a distal sequence element (DSE).

[0151] An "enhancer" is a DNA sequence that can stimulate transcriptional activity, and may be a native element of a promoter or a heterologous element that enhances the level or tissue specificity of expression. An "enhancer" can act in either orientation (5'->3' or 3'->5') and may function when placed either upstream or downstream of a promoter.

[0152] Promoters and / or enhancers may be derived in their entirety from a native gene, may be composed of different elements derived from different elements found in nature, or may even be composed of synthetic DNA segments. A promoter or enhancer may contain DNA sequences involved in binding protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological conditions, or developmental conditions.

[0153] Non-limiting examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, polII promoter, polIII promoter, synthetic promoter, hybrid promoter, etc. Furthermore, sequences derived from non-viral genes, such as mouse metallothionein gene, are also used herein. Exemplary constitutive promoters include promoters of the following genes that code for certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6, and other constitutive promoters known to those skilled in the art. Furthermore, many viral promoters function constitutively in eukaryotic cells. These include, inter alia, 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. Thus, any of the above constitutive promoters can be used to control the transcription of the heterologous gene insert.

[0154] "Transgene" is used herein to refer to a nucleic acid sequence / polynucleotide that is conveniently intended or introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene, that encodes an inhibitory RNA or a polypeptide or protein, and is generally heterologous to the native AAV genome sequence.

[0155] The term "transducing" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Thus, the introduction of a transgene into a cell by a viral particle may be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. Once the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and further inherited or inherited by the descendant cells or organisms of the recipient cell or organism. Finally, the introduced transgene may be present extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced via transduction. Thus, a "transduced" cell is a cell into which a transgene has been introduced, or a descendant thereof. The transduced cell can proliferate, transcribe the transgene, and express the encoded inhibitory RNA or protein. For gene therapy uses and methods, the transduced cell may be in a mammal.

[0156] A transgene under the control of an inducible promoter is expressed only in the presence of an inducing agent or is expressed to a greater extent in the presence of the inducing agent (e.g., transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters contain a response element (RE) that stimulates transcription when an inducing factor binds. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. To obtain an inducible response, promoters containing a particular RE can be selected, and in some cases, the RE itself can be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive vs. inducible; strong vs. weak), both the presence and expression level of the polypeptide can be controlled in the genetically modified cells. If the gene encoding the polypeptide is under the control of an inducible promoter, in situ polypeptide delivery can be triggered by exposing the genetically modified cells in situ to conditions that allow for polypeptide transcription, for example, by intraperitoneal injection of a particular inducer of the inducible promoter that controls transcription of a drug. For example, in situ expression by genetically modified cells of a polypeptide encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically modified cells in situ with a solution containing the appropriate (i.e., inducible) metal ion.

[0157] A nucleic acid / transgene is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. The nucleic acid / transgene encoding an RNAi or polypeptide, or the nucleic acid inducing the expression of a polypeptide, may include an inducible promoter or a tissue-specific promoter to control the transcription of the encoded polypeptide. The nucleic acid that is operably linked to an expression control element may also be called an expression cassette.

[0158] In certain embodiments, cell type specific or inducible promoters, enhancers, etc. are used in the methods and uses described herein. Non-limiting examples of cell type specific promoters include those isolated from genes from TMC1, TMC2, Espin, PCDH15, PTPRQ, TMHS (LHFPL5), MYO1A. Non-limiting examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and IFN.

[0159] In certain embodiments, the expression control element comprises a CMV enhancer. In certain embodiments, the expression control element comprises a β-actin promoter. In certain embodiments, the expression control element comprises a chicken β-actin promoter. In certain embodiments, the expression control element comprises a CMV enhancer and a chicken β-actin promoter.

[0160] The term "modify" or "variant" and its grammatical variants as used herein mean that a nucleic acid, polypeptide, or subsequence thereof deviates from a reference sequence.Thus, modified and variant sequences may have substantially the same, greater or less expression, activity, or function as the 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 that has a mutation, for example, a missense mutation or a nonsense mutation.

[0161] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that is genetically altered compared to the wild type. The sequence may be genetically altered without altering the encoded protein sequence. Or, the sequence may be genetically altered to encode a variant protein. A nucleic acid or polynucleotide variant may also refer to a combination of sequences that are codon-altered to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild type protein sequence, and that are codon-altered to encode a variant protein. For example, some codons of such nucleic acid variants are altered without altering the amino acid of the protein encoded thereby, and some codons of nucleic acid variants are altered, resulting in altered amino acid of the protein encoded thereby.

[0162] The terms "protein" and "polypeptide" are used interchangeably herein. A "polypeptide" encoded by a "nucleic acid" or "polynucleotide" or "transgene" disclosed herein includes partial or full-length native sequences as well as native 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. Thus, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences need not be identical to endogenous proteins that are defective or whose activity, function, or expression is insufficient, missing, or absent in the mammal being treated.

[0163] 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).

[0164] An example of an amino acid modification is a conservative amino acid substitution or deletion. In certain embodiments, the modified or variant sequence retains at least some of the function or activity of the unmodified sequence (e.g., the wild-type sequence).

[0165] Another example of an amino acid modification is a targeting peptide introduced into the capsid protein of the viral particle. Peptides have been identified that target recombinant viral vectors to the central nervous system, for example to distinct brain regions.

[0166] Such modified recombinant viruses may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, recombinant viruses having modified capsid proteins may "target" cerebrovascular epithelial tissue by binding at a higher level than a comparable, unmodified capsid protein. For example, recombinant viruses having modified capsid proteins may bind to cerebrovascular epithelial tissue at a level 50%-100% higher than unmodified recombinant viruses.

[0167] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in most organisms is the genetic material, whereas ribonucleic acid (RNA) is responsible for transferring the information contained in DNA to proteins. Fragments and variants of the disclosed nucleotide sequences, as well as proteins encoded thereby or partial length proteins, are also encompassed by the present invention. By "fragment" or "portion" is meant a full-length or less than full-length nucleotide sequence encoding a polypeptide or protein, or a full-length or less than full-length 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 the wild-type activity or function).

[0168] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the naturally occurring molecule. In the case of a nucleotide sequence, the variant includes sequences that, due to the degeneracy of the genetic code, code for the same amino acid sequence of the naturally occurring protein. Naturally occurring allelic variants, for example, these variants can be identified using molecular biology techniques, for example, using polymerase chain reaction (PCR) and hybridization methods. Variant nucleotide sequences also include synthetically obtained nucleotide sequences, such as nucleotide sequences generated using site-directed mutagenesis, that code for naturally occurring proteins, as well as nucleotide sequences that code for polypeptides with amino acid substitutions. Typically, a nucleotide sequence variant of the invention will have at least 40%, 50%, 60% to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, typically 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 variants are biologically functional (i.e., retain 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0169] A "conservative variation" of a particular nucleic acid sequence refers to a nucleic acid sequence that codes for the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given polypeptide. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, at every position where arginine is specified by a codon, this codon can be changed to any of the corresponding codons described without changing the encoded protein. Such nucleic acid variations are "silent variations," which are a type of "conservatively modified variations." All nucleic acid sequences described herein that code for a polypeptide also represent all possible silent variations, unless otherwise specified. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is usually the only codon for methionine) can be modified by standard techniques to produce a functionally identical molecule. Thus, each "silent variation" of a nucleic acid that codes for a polypeptide is implied in each described sequence.

[0170] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having 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 with standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For these purposes, substantial amino acid sequence identity typically means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0171] The term "substantial identity" in the context of a polypeptide indicates that the polypeptide comprises a sequence having 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 a reference sequence over a specified comparison window. Identical sequences of two polypeptides indicate that a polypeptide is immunologically reactive with an antibody raised against the second polypeptide. Thus, for example, a polypeptide is identical to a second polypeptide when the two polypeptides differ only in conservative substitutions.

[0172] "Mechanosensation" means a response to a mechanical stimulus. Touch, hearing, and balance are examples of the conversion of mechanical stimuli into neuronal signals. Mechanosensory input is converted into a response to a mechanical stimulus through a process called "mechanotransduction."

[0173] "Disease" refers to 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 loss of function of a protein that functions in mechanosensory transduction, for example, expressed 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, the disease is hearing loss associated with a genetic defect, for example, a defect in TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHKN, CLRN1, PDZD7, USH1C (e.g., harmonin-a, b, or c).

[0174] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, inhibit, reduce, or diminish undesirable physiological changes or disorders, such as the development, progression, or worsening of a disorder. Beneficial or desirable clinical results for purposes of the present invention include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of disease symptoms or side effects (i.e., no worsening or progression), delay or slowing of disease progression, remission or alleviation, and remission (partial or complete), whether detectable or undetectable. "Treatment" can also mean extending survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a condition or disorder, and those who are predisposed to the disease (e.g., as determined by genetic assays).

[0175] As used herein, "essentially free" is used herein to mean that, in terms of the named components, none of the named components are intentionally formulated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the named components resulting from unintentional contamination of the composition is significantly less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the named components cannot be detected using standard analytical methods.

[0176] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0177] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to mutually exclusive alternatives, but this disclosure supports the definition referring to alternatives only and "and / or." As used herein, "another" can mean at least a second, or more.

[0178] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of the device, the inherent variation of the method used to determine the value, the variation that exists between study subjects, or a value that is within 10% of the stated value.

[0179] VI. Kit The present invention provides kits comprising packaging material and one or more components therein. The kits typically comprise a label or insert containing a description of the components or instructions for using the components therein in vitro, in vivo, or ex vivo. The kits may comprise a collection of such components, e.g., nucleic acids, recombinant vectors, and / or viral particles.

[0180] A kit refers to a physical structure that contains one or more components of the kit. The packaging material is capable of maintaining the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0181] The label or insert may include the identity of one or more components therein, the dose, the mechanism of action, the pharmacokinetics, and the clinical pharmacology of the active components, including pharmacokinetics. The label or insert may include information identifying the manufacturer, the lot number, the place and date of manufacture, the expiration date. The label or insert may include information identifying the manufacturer information, the lot number, the place and date of manufacture. The label or insert may include information regarding the disease for which the components of the kit may be used. The label or insert may include instructions to the clinician or subject for the method, use, or use of one or more of the components of the kit in a treatment protocol or therapeutic regimen. The instructions may include dosage, frequency, or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0182] The label or package insert may include information about any benefit that the component may provide, such as a preventive benefit or a therapeutic benefit. The label or package insert may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which it may be inappropriate to use a particular composition. Adverse side effects or complications may also occur when the subject takes, plans to take, or is currently taking one or more other drugs that may be incompatible with the composition, or when the subject has, plans to take, or is currently taking another treatment protocol or therapeutic regimen that may be incompatible with the composition, and therefore the instructions may include information about such incompatibilities.

[0183] A label or package insert includes "printed matter", e.g., paper or cardboard, separate from or affixed to the component, kit, or packaging material (e.g., box), or attached to the ampoule, tube, or vial containing the kit component. A label or package insert may further comprise a computer readable medium, e.g., a printed label with a bar code, a disk, an optical disk, e.g., CD- or DVD-ROM / RAM, DVD, MP3, or an electronic storage medium, e.g., RAM and ROM or hybrids thereof, e.g., magnetic / optical storage media, FLASH memory, hybrids, and memory type cards. EXAMPLES

[0184] VII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by one of skill in the art that the techniques disclosed in the following examples demonstrate techniques discovered by the inventors to function well in the practice of the invention, and therefore are believed to constitute preferred embodiments for practicing the invention. However, in light of this disclosure, it should be understood by one of skill in the art that many changes can be made in the specific embodiments disclosed and still obtain a similar or similar result without departing from the spirit and scope of the invention.

[0185] Example 1 - Delivery of AAV vectors to the cochlea Gene therapy is a powerful tool to combat hearing loss and hearing loss, but its clinical implementation is hindered by the anatomy of the temporal bone, the high diversity of genetic lesions, and the myriad of cochlear sensory and supporting cell types. With the identification of adeno-associated virus (AAV) capsids capable of efficient hair cell transduction, including Anc80L65 and AAV2, cochlear gene therapy for several types of genetic hearing loss has come closer to clinical realization. However, for genetic lesions that affect hearing at the level of cochlear supporting cells, additional vectors with different tropism properties are required. To address this need, we performed an AAV directed evolution screen in non-human primate (NHP) cochleae to identify an AAV capsid variant library with diverse transduction properties that can meet the requirements of current and future cochlear gene therapy designs.

[0186] Peptide-modified AAV libraries were generated by random peptide insertion into AAV1, AAV2, and AAV9, respectively, by random sequence insertion into position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, and position 588 of the AAV9 capsid. Cochlea-specific AAV enrichment studies were performed in which the AAV capsid library (3E11vg) was delivered directly into the cochlea by the same RWM+LCF method. An eGFP expression construct was packaged into AAV driven by the CAG promoter. The AAV capsid library was injected into four cochleae (two NHPs) over the course of two rounds of in vivo enrichment.

[0187] NGS amplicon sequencing libraries were generated for each enrichment round, and the enrichment of each capsid configuration was tracked using the sequencing data. First, AAV capsid libraries were delivered to NHP brains by intracerebroventricular (ICV) injection. In this experiment, a significant and unexpected transduction of NHP cochlear hair cells and supporting cells by AAV9 was observed. This unexpected transduction of cochlear cells after ICV injection is likely due to the fluid connection between CSF and perilymph via the perilymphatic duct.

[0188] Enrichment heatmaps were generated for each round. These illustrate the enrichment of the indicated barcodes at baseline (round 0) and after rounds 1 and 2 of in vivo passage in rhesus macaques. To generate these, fastq result files for each tissue and round combination were processed with custom Python scripts designed to extract and quantify the unique barcode configurations observed at the DNA level. A custom R script was used to calculate the percent of barcodes present in each sample and convert DNA barcodes to amino acid barcodes. Figure 6 corresponds to samples treated with AAV1-derived libraries. Figure 8 corresponds to samples treated with AAV2-derived libraries. Figure 10 corresponds to samples treated with AAV9-derived libraries.

[0189] Wild-type and modified AAV vectors were successfully delivered to the cochlea of ​​rhesus monkeys. AAV vectors can transduce cochlear inner hair cells and a collection of supporting cells including cells of the stria vascularis and spiral ganglion neurons. Sequencing results from cochlear AAV enrichment revealed a collection of enriched capsid variants, which are undergoing small library and fluorescence-based validation.

[0190] Example 2 - Therapeutic delivery to the cochlea via CSF There is a need for non-invasive and non-surgical methods to facilitate cochlear therapeutic delivery. Herein, we report that robust cochlear delivery was achieved by ICV delivery of AAV through the CSF in rhesus monkeys and mice. This CSF-based cochlear therapeutic delivery method is facilitated by diffusion of therapeutic particles through the perilymphatic duct, a non-occlusive fluid connection of the CSF in the brain, into the perilymph-filled scala vestibuli and scala tympani of the cochlea. This finding opens the door to the use of the CSF route to deliver human cochlear therapeutics in the clinical setting.

[0191] In a clinical setting, it may be advantageous to deliver AAV or other therapeutic agents for cochlear transduction to the CSF via lumbar puncture, a non-surgical outpatient procedure that can be completed in minutes.

[0192] In clinical settings, it may be advantageous to deliver AAV or other therapeutic agents for cochlear transduction to the CSF via intracerebroventricular (ICV) injection. For this procedure, a hole is drilled in the skull and an injection needle is precisely inserted to target the ventricular space. This procedure is more invasive than a lumbar puncture, but less invasive than some middle ear surgical approaches.

[0193] In a clinical setting, it may be advantageous to deliver AAV for cochlear transduction or other therapeutic agents to the CSF via other methods that reach the CSF space and facilitate diffusion of the AAV capsid or other therapeutic agent to the cochlea.

[0194] In clinical settings, it may be advantageous to deliver AAV for cochlear transduction or other therapeutic agents to the CSF via the intracisternal or common intrathecal space, which would reach the CSF space and facilitate diffusion of the AAV capsid or other therapeutic agent to the cochlea.

[0195] Example 3 - AAV evolution after direct cochlear injection To assess the enrichment of AAV capsid candidates across successive in vivo capsid library evolution rounds, the UMI percent contribution from each capsid barcode was visualized as a heatmap (Figure 6, Figure 8, and Figure 10). In each heatmap, from left to right, the indicated columns are DNA extracted from round 1, DNA extracted from round 2, and RNA extracted from round 2. It can be seen that the percent contribution of the indicated capsid candidates increases from column 1 (round 1 DNA) to column 2 (round 2 DNA). Detection of high levels of capsids in round 2 RNA samples (column 3) varied among the top-ranked round 2 DNA capsid candidates. Both DNA and RNA performance are informative indicators of capsid candidate transduction properties, and therefore care was taken to select validation capsid candidates that performed well in both round 2 DNA and RNA categories. Among the AAV1-derived capsid candidates, capsid candidate LGGSAAR (SEQ ID NO:39) was selected due to its strong DNA and RNA performance in both the cochlea and pooled vestibular tissues (Figure 6). Capsid candidate KAGGSQG (SEQ ID NO:84) was selected due to its strong DNA and RNA performance in the cochlea, despite its low RNA performance in vestibular tissues (Figure 8). No capsid candidates were selected from AAV9 because only a limited number of fluorescent validation positions (8) were available and other criteria were also considered (Figure 10).

[0196] Additional capsid candidates were selected based on the average UMI counts as well as the rank order of fold enrichment of % Round 2 DNA UMI counts relative to the % DNA UMI counts obtained from the Round 2 input vector pool (Figure 12).

[0197] To select additional capsid candidates with favorable round 2 detection:round 2 input ratios, the distribution of all capsid candidates was visualized using a scatter plot method. In general, candidates that are highly abundant in the round 2 input capsid library tend to be the most highly detected in the round 2 output library by raw UMI counts. After normalization, the most enriched capsids also tend to be the most under-detected capsids in the round 2 input capsid library. Some candidates with low, but not minimal, round 2 input levels that were also robustly enriched were also selected. For this purpose, promising outlier candidates were identified from the scatter plot distribution. Using this method, no capsid candidates were selected from AAV1 (Figure 13A). However, using this method, the AAV2-derived candidate AAKVAAP (SEQ ID NO: 154) (Figure 13B) and the AAV9-derived candidate RSGVGSA (SEQ ID NO: 155) (Figure 13C) were selected.

[0198] All capsid candidates selected for progression to fluorescent validation are listed in Figure 14A along with the parent serotype indicated and a description of the criteria used to select the capsid. Capsids were grouped into two validation pools to facilitate delivery of separate fluorescently tagged capsids, one to each ear of a non-human primate (Figure 14B). Capsid candidates were individually generated to deliver a fluorescent reporter expression construct. Capsids were pooled in groups of four to create validation pools. One validation pool is delivered by direct intracochlear injection into each ear of a rhesus monkey. Thirty days after injection, animals are sacrificed and cochleae are harvested for histological evaluation (Figure 14C).

[0199] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions 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.

[0200] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024517710000002.tif93150

Claims

1. A modified adeno-associated virus (AAV) containing a therapeutic transgene operably linked to a cochlear-specific promoter.

2. The modified AAV of claim 1, wherein the therapeutic transgene treats or prevents hearing loss or vestibular disorders.

3. The modified AAV of claim 1, wherein the cochlea-specific promoter is a hair cell-specific promoter.

4. The modified AAV of claim 1, wherein the cochlea-specific promoter is a supporting cell-specific promoter, such as the GJB2 promoter.

5. The modified AAV of any one of claims 1 to 4, comprising a modified capsid protein.

6. The modified AAV of claim 5, wherein the modified capsid protein comprises a targeting peptide, and the targeting peptide is 3 to 10 amino acids in length.

7. The modified AAV of claim 5, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

8. The modified AAV of claim 7, wherein the modified AAV capsid protein is derived from the AAV1 capsid protein and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

9. The modified AAV of claim 8, wherein the targeting peptide is selected from SEQ ID NOs: 150, 151, and 1-44.

10. The modified AAV of claim 9, wherein the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length.

11. The modified AAV of claim 10, wherein the linker sequence is SSA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide.

12. The modified AAV of claim 7, wherein the modified AAV capsid protein is derived from the AAV2 capsid protein and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

13. The modified AAV of claim 12, wherein the targeting peptide is selected from SEQ ID NOs: 152, 154, and 45-100.

14. The modified AAV of claim 13, wherein the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length.

15. The modified AAV of claim 14, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide.

16. The modified AAV of claim 7, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

17. The modified AAV of claim 16, wherein the targeting peptide is selected from SEQ ID NOs: 153, 155, and 101-149.

18. The modified AAV of claim 17, wherein the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length.

19. The modified AAV of claim 18, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide.

20. The modified AAV of claim 6, wherein the targeting peptide is 7 amino acids in length.

21. A pharmaceutical composition comprising the modified AAV of any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

22. The pharmaceutical composition of claim 21 for use in a method of delivering a therapeutic transgene to the cochlea of ​​a subject to treat or prevent hearing or vestibular disorders.

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