US20200101122A1

US20200101122A1 - Method for treating an auditory neuropathy spectrum disorder

Info

Publication number: US20200101122A1
Application number: US16/586,768
Authority: US
Inventors: Yen-Fu Cheng; Chen-Chi Wu; Ying-Chang LU
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-27
Filing date: 2019-09-27
Publication date: 2020-04-02
Also published as: WO2020069320A4; WO2020069320A1; TW202027778A; EP3856227A1; US20220000972A1; EP3856227A4

Abstract

The present invention provides a method for treating a method for treating an auditory neuropathy spectrum disorder in a subject comprising transferring a transgene via an adeno-associated virus (AAV) vector to the subject; wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, AND OTOF.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/737,406, filed on Sep. 27, 2018, all of which are hereby expressly incorporated by reference into the present application.

FIELD OF THE INVENTION

The present invention relates to a method for treating an auditory neuropathy spectrum disorder.

BACKGROUND OF THE INVENTION

Hearing loss is the most common pediatric sensory defect: more than 1/1000 children are affected by severe to profound sensorineural hearing impairment (SNHI) [1]. Pediatric SNHI is composed of a plethora of disease entities. Among them, auditory neuropathy spectrum disorder (ANSD) is of special interest because of its unparalleled clinical manifestations. ANSD is not uncommon, accounting for approximately 7% of permanent childhood hearing loss and a significant (but as yet undetermined) proportion of adult impairment [2]. Patients with ANSD have various degrees of hearing loss with poor speech perception that is out of proportion to their hearing levels [3]. Audiologically, ANSD is characterized by the preservation of normal outer hair cell function as evidenced by the presence of otoacoustic emissions (OAEs) and/or cochlear microphonics (CM), whereas the transmission of the auditory signal to the brainstem is impaired as evidenced by abnormal sound-evoked potentials of auditory brainstem response (ABR), poor speech perception and the absence of acoustic reflexes [3-5]. The pathophysiology of ANSD has been proposed to involve an abnormal peripheral auditory system localized to the inner hair cells, the auditory nerve, or the synapse between them [6]. Etiologically, ANSD might be caused by environmental insults, including infection during pregnancy, prematurity, perinatal hypoxemia and neonatal hyperbilirubinemia [7, 8], or it might be the consequence of certain syndromes, such as Charcot-Marie-Tooth disease [9] or cri-du-chat syndrome [10]. The tendency of familial aggregation observed in some series suggests that genetic factors may also be involved in the pathogenesis [6-8]. It has been estimated that approximately 40% of ANSD cases may have a genetic basis [11].
It is desirable to develop a new method for treating an auditory neuropathy spectrum disorder.

BRIEF SUMMARY OF THE INVENTION

It was unexpectedly discovered in the present invention that an auditory neuropathy spectrum disorder can be efficiently treated through a gene therapy via a vector comprising an Adeno-associated virus (AAV), called as an AAV vector hereinafter.
The present invention provides a method for treating a method for treating an auditory neuropathy spectrum disorder in a subject comprising transferring a transgene via an adeno-associated virus (AAV) vector to the subject; wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.
In another aspect, the present invention provides a construct for delivering a transgene to a subject suffering from an auditory neuropathy spectrum disorder, which comprises an adeno-associated virus (AAV) and the nucleic acids designated a transgene; wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.

The drawings presenting the preferred embodiments of the present invention are aimed at explaining the present invention. It should be understood that the present invention is not limited to the preferred embodiments shown. The data in the figures and examples are shown as mean±standard deviation (SD). Significant differences are shown as follows: *p<0.05, ***p<0.001.

FIG. 1 provides the genes identified to be associated with good and poor CI outcomes.

FIG. 2 provides an image of the auditory thresholds of Pjvk^WT/WTand Pjvk^G292R/G292Rmice. FIG. 2(A) shows that ABR thresholds were measured in 3-week-old and 6-week-old mice. Pjvk^G292R/G292Rmice (red) showed progressive severe hearing loss as compared to Pjvk^WT/WTmice (blue) at all frequencies (n=10 for each group; thresholds expressed in mean±SD). FIG. 2(B) shows that ABR traces (clicks-stimuli) at 100 dB SPL were superimposed (Pjvk^WT/WT, black; Pjvk^G292R/G292R, red). Note that wave I-V in 3-week-old Pjvk^G292R/G292Rmice showed increased latencies and reduced peak amplitudes. FIG. 2(C) shows the patterns of parvalbumin immunolocalization in hair cells and spiral ganglion neurons of Pjvk^G292R/G292Rmice. Degeneration of organ of Corti and spiral ganglion neurons are indicated by arrows and circles, respectively. (Bar=50 μm).

FIG. 3 shows the construct of an AAV vector containing the gene—Pjvk.

FIG. 4 shows the ABR thresholds at 8, 16, and 32 kHz in Anc80L65.Pjvk—treated versus untreated Pjvk^G292R/G292Rmice.

FIG. 5 provides the results of the Anc80-Pjvk gene therapy, showing that the circling behavior was reduced in treated mice.

FIG. 6 provides the results of the Anc80-Pjvk gene therapy, showing that the rotarod performance was improves in treated mice.

FIG. 7 provides the results of the Anc80-Pjvk gene therapy, showing that the swimming performance was improves in treated mice.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

1. Non-Syndromic Auditory neuropathy spectrum disorder (ANSD)

Hearing loss is the most common pediatric sensory defect: more than 1/1000 children are affected by severe to profound sensorineural hearing impairment (SNHI) [1]. Pediatric SNHI is composed of a plethora of disease entities. Among them, auditory neuropathy spectrum disorder (ANSD) is of special interest because of its unparalleled clinical manifestations. ANSD is not uncommon, accounting for approximately 7% of permanent childhood hearing loss and a significant (but as yet undetermined) proportion of adult impairment [2]. Patients with ANSD have various degrees of hearing loss with poor speech perception that is out of proportion to their hearing levels [3]. Audiologically, ANSD is characterized by the preservation of normal outer hair cell function as evidenced by the presence of otoacoustic emissions (OAEs) and/or cochlear microphonics (CM), whereas the transmission of the auditory signal to the brainstem is impaired as evidenced by abnormal sound-evoked potentials of auditory brainstem response (ABR), poor speech perception and the absence of acoustic reflexes [3-5]. The pathophysiology of ANSD has been proposed to involve an abnormal peripheral auditory system localized to the inner hair cells, the auditory nerve, or the synapse between them [6].
Etiologically, ANSD might be caused by environmental insults, including infection during pregnancy, prematurity, perinatal hypoxemia and neonatal hyperbilirubinemia [7, 8], or it might be the consequence of certain syndromes, such as Charcot-Marie-Tooth disease [9] or cri-du-chat syndrome [10]. The tendency of familial aggregation observed in some series suggests that genetic factors may also be involved in the pathogenesis [6-8]. It has been estimated that approximately 40% of ANSD cases may have a genetic basis [11].
The hearing loss levels in patients with ANSD vary from mild to profound hearing loss, and their speech perception may be out of proportion to the audibility changes. In addition, patients with auditory neuropathy do not typically derive much benefit from hearing aids. Some of the patients are able to acquire speech and hearing without a hearing aid over time; some of them present well with hearing aids or cochlear implants (CIs); and still a part of them did not develop well in speech or hearing despite under CI use. These features lead to difficulties in the diagnosis and treatment for patients with ANSD in clinical practice.

2. Pjvk Mutation and Non-Syndromic ANSD

Mutation in Pjvk is a common cause of non-syndromic ANSD in humans. Delmaghani et al. identified mutations in the Pjvk gene in four familial cases of ANSD [12]. Two missense mutations were identified in the families. The Pjvk gene produces a protein the researchers named “pejvakin”, which is expressed in the organ of Corti, the spiral ganglion and the neuronal cell bodies of the cochlear nuclei, superior olivary complex and the inferior colliculus of the afferent auditory pathway. The researchers believe pejvakin is crucial for auditory nerve signalling. Mutation in this gene appears to result in auditory neuropathy due to a disruption in neuronal signalling along the auditory pathway. Cochlear function is intact in these patients.
The PCDH15 gene is a member of the cadherin superfamily. Family members encode integral membrane proteins that mediate calcium-dependent cell-cell adhesion. it plays an essential role in maintenance of normal retinal and cochlear function. Mutations in this gene result in hearing loss and Usher Syndrome Type IF (USH1F). Extensive alternative splicing resulting in multiple isoforms has been observed in the mouse ortholog.

3. Gene Mutation and Poor CI Outcome

We identified patients with mutations in some genes, including Pjvk, PCDH15 GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF, usually exhibit excellent long-term CI outcomes, probably because the effects of these mutations were confined to the inner ear and the function of the auditory nerve is spared. Based on the results of the clinical trial in multi-center studies for the purpose to identify the genetic determinants of poor CI outcomes in Taiwan, which enrolled more than 300 children with CIs, we identified genetic variants which are associated with poor CI outcomes in 7 (58%) of the 12 cases; 4 cases had bi-allelic PCDH15 pathogenic mutations and 3 cases were homozygous for the Pjvk p.G292R variant. Mutations in the WFS1, GJB3, ESRRB, LRTOMT, MYO3A, and POU3F4 genes were detected in 7 (23%) of the 30 matched controls. The allele frequencies of PCDH15 and Pjvkvariants were significantly higher in the cases than in the matched controls (both P<0.001). In the 7 CI recipients with PCDH15 or Pjvkvariants, otoacoustic emissions were absent in both ears, and imaging findings were normal in all 7 implanted ears. PCDH15 or Pjvkvariant is associated with poor CI performance, yet children with PCDH15 or Pjvkvariants might show clinical features indistinguishable from those of other typical pediatric CI recipients.
4. Adeno-Associated Virus (AAV)
The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies showed an efficient transduction of sensory cells in adult ears while maintaining normal cochlear functions [13].
Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, Luk H. Vandenberghe labortory inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications [14]. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina. Recently, novel adeno-associated virus (AAV) serotypes, such as Anc80, have been confirmed as a promising delivery system for restoring the function of inner ear sensory cells [15]. However, the efficiency of these new AAVs in targeting other pathological changes of the auditory/vestibular pathways remains unclear.
In one preferred embodiment of the invention, the AAV vector is an AAV vector comprising an Anc80 capsid protein as provided in WO2017/100791 A1, also called as an AAV-Anc80 vector. The AAV-Anc80 vector was confirmed to be able to efficiently deliver nucleic acids to the inner ear, e.g., cochlea, particularly the inner and outer hair cells (IHCs and OHCs) in the cochlea, which is an attractive target for gene therapy approaches to intervene in hearing loss and deafness of various etiologies, most immediately monogenic forms of inherited deafness.
In one more preferred embodiment of the invention, the AAV vector is a synthetic inner ear hair cell targeting adeno-associated virus (AAV) vector, wherein the vector encodes a capsid having at least about 85% sequence identity to Anc80, and comprises a promoter selected from the group consisting of an Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter that directs expression of harmonin-a, harmonin-b, or harmonin-c polypeptide, as provided in WO2018/145111 A1.
5. Gene Therapy
We have completed genetic studies in more than 250 patients with cochlear implantation. Mutations in most genes are related to good CI outcomes, but mutations in PCDH15 and Pjvk are associated with unfavorable CI outcomes (see FIG. 1). Animal studies are currently underway to elucidate the pathogenetic mechanisms and to explore novel therapeutic approaches. Some genes have been identified to be associated with good and poor CI outcomes, as shown in FIG. 1.
The invention is further illustrated by the following example, which should not be construed as further limiting.

Example

1. Animal Model
Genome-editing techniques were used to produce Pjvk p.G292R gene transgenic mice, in which the hearing characterization has been completed in the PjVk^G292R/G292Rmouse with the c.874 G>A mutation. Pjvk^G2921/G292Rmice showed significantly higher hearing thresholds than PjVk^G292R/WTand Pjvk^WT/WTmice at clicks, as well as 8 k, 16 k and 32 kHz pure tones on auditory brainstem response (FIG. 2A). Further analysis of the waveforms revealed prolonged latencies of all the ABR waves (FIG. 2B), indicating the presence of retrocochlear lesions. In accordance with the audiological findings, pathological changes in hair cells and spiral ganglion neurons were observed in PjVkG^2921/G292Rmice at 1 Month (FIG. 2C). In addition to the hearing phenotypes, Pjvk^G2921/G292Rmice also demonstrated equilibrium deficits suggesting pathologies involving both the vestibular organ (circling) and the central nervous system (nodding).
2. Gene Therapy
The full length sequence of Pjvk gene was cloned into the AAV expression vector. The construct can be co-transfected with the AAV-helpler plasmid (Anc80L65) into the HEK293 cells and produced the Anc80 virus particles. The construct of AAV-Pjvk was shown in FIG. 3.
A nanoliter microinjection system (Nanoliter2000; World Precision Instruments) was used to load Anc80L65.Pjvk into the glass micropipette (10 mm in diameter). A total of 0.7 ul Anc80L65.Pjvk was injected into round window membrane of inner ear of Pjvk p.G292R mice at P0-P3. Sham surgeries were performed as above with Anc80L65-GFP as a negative control virus. At P45, the treated mice showed lower auditory brainstem response thresholds and improved vestibular function as compared to the control group. The findings suggested that the Anc80-directed gene therapy was an efficient delivery system for simultaneously introducing genes to both the sensory cells of the inner ear and the neurons of the central auditory/vestibular pathways.
The Anc80 virus particles were injected into the round window membrane by the microinjection.
3. Audiological Evaluations
Mice were anesthetized with sodium pentobarbital (35 mg/kg) delivered intraperitoneally and maintained in a head-holder within an acoustically and electrically insulated and grounded test room. We used an evoked potential detection system (Smart EP 3.90, Intelligent Hearing Systems, Miami, Fla., USA) to measure the thresholds of the auditory brainstem response (ABR) in mice. Click sounds as well as 8, 16, and 32 kHz tone bursts at varying intensity (from 10 to 130 dB SPL), were given to evoke ABRs of mice. The response signals were recorded with a subcutaneous needle electrode inserted ventrolaterally into the ears of the mice.
The results of the gene therapy were shown in FIG. 4, showing that the cochlear function was rescued. The Pjvk^G2921/G292Rmice treated with an AAV vector comprising Anc80 (Anc80-Pjvk) provided lower ABR thresholds measured at 6 weeks showed improved hearing thresholds in the treated ears as compared with the untreated mice, especially at 8, 16 and 32 kHz.
4. Vestibular Evaluations
Mice were subjected to a battery of vestibular evaluations, including observation of their circling behavior and head-tilting (performed at P90-120), a reaching test, a swimming test, and a rotarod test (all performed at P90-120 age). The reaching responses of mice were recorded after suspending animals by their tails and observing the reaction of their limbs and head-bobbing behaviors. Mice that extended their forelimbs and tried to reach a surface were considered normal (i.e., in terms of reaching response), whereas animals that either clasped their forelimbs or exhibited head-bobbing behavior were classified as abnormal. For the swimming test, mice were observed for 15-20 s, and those that maintained themselves well at the water surface were classified as normal, whereas those that failed to stay near the surface were considered abnormal. For the gripping test, mice were placed on the lower end of a 45-cm-long metal stick, and those that required more than 15 s to reach the top of the stick were classified as abnormal (i.e., <15 s were normal). For the rotarod test, mice were assessed for their ability to balance on a revolving rod (i.e., rotarod) of 3.5 cm diameter. For each test, the mouse was placed on the rod rotating at 35 rpm, and the time required for the mouse to fall was recorded. Each mouse was tested 5 times, and the results were averaged.
5. Circling Behavior
The circling behavior of mice was quantified using optical tracking. A 38-cm×58-cm box was attached to a video camera (gopro). The ImageJ software was set to track the head of mice placed within the box. Each mouse was placed into the box and allowed to acclimate to the new environment for 2 min. Complete rotations were recorded and quantified for the next 2 min, followed by a 1-min “cool-down” period in which rotations were not tracked. Each mouse was assessed three times on the same day, and the average was taken.
As shown in FIG. 5, the Pjvk mutant mice had significant vestibule dysfunction, as evidenced by their circling behavior. It was confirmed that Anc80-Pjvk gene therapy reduced circling behavior in the mice treated with the gene therapy. After the gene therapy, the P90-120 mice was sent to the 30 sec-duration tracking of treated mice and untreated (mutant) control mice. The untreated mice had developed an obvious circling behavior and showed poor ability to reach the field boundary (FIG. 5, left). On the contrary, the treated mice did not show the circling behavior and could arrive the most boundary of the testing field without the interruption of moving direction by circling (FIG. 5, right).
It was also confirmed in the rotarod test that the gene therapy improved the balance function in rotarod test. The rotarod test is a measure of balance function in which mice are placed on a rod that rotates with increasing velocity, and the length of time the mouse remaining balanced on the rotating rod is recorded. The Pjvk mutant mice also showed diminished balance on the rotarod test. After gene therapy, the treated mice exhibited a better ability to stay on the rotating rod, and the untreated control mice showed a poor ability to stay on the rod with a shorter retaining time (65.1±13.1 sec) than the treated mice (213.4 ±12.3 sec), see Table 1 and FIG. 6.

TABLE 1

The result of rotarod test at P90~120

	Pjvk	Pjvk	Pjvk
Normal	untreated	treated w/	treated w/
control	control	Anc80-Pjvk	Anc80-GFP
(n = 6)	(n = 8)	(n = 8)	(n = 5)

Time on	213.2 ± 7.2	65.1 ± 13.1	213.4 ± 12.3	79.4 ± 7.7
rod (sec)

6. Swim Testing
Mice were placed in a large container filled with room temperature water. Their swimming behavior was recorded using a video camera over 2 min. The videos were de-identified and scored by an observer who was blinded to the genotype and treatment and who was also not involved with the initial video recording. A well-established 0-3 scoring system was used to assess the swim performance. Briefly, a score of 0 indicates normal swimming behavior. A score of 1 indicates mild swimming abnormality (circling, vertical swimming). A score of 2 indicates moderate swimming abnormality (immobile floating). A score of 3 indicates significant swimming abnormality in which the mouse needs to be rescued immediately (underwater tumbling). Swimming testing was performed at P90˜120 in all animals (6 normal control mice, 8 untreated control mice, 8 mutant mice that received Anc80-Pjvk gene therapy and 5 mutant mice that received Anc80-GFP), see FIG. 7.
7. Inner Ear Morphology Studies
Tissues from inner ears of mice were subjected to hematoxylin and eosin (H&E) staining, and the morphology of each sample was examined with a Leica optical microscope. For light microscopy, inner ears from adult mice were fixed by perilymphatic perfusion with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) through round and oval windows and a small fenestra in the apex of the cochlear bony capsule. Specimens were subsequently rinsed in PBS buffer and decalcified in 4% PFA with 0.35 M EDTA at 4° C. for 1 week. For light microscopy studies, samples were dehydrated and embedded in paraffin. Subsequently, serial sections (7 μm) were stained with H&E.
Whole-mount studies of mouse inner ear specimens were performed as previously described
with some minor modification. Briefly, after perfusion with 4% PFA, the cochleae were postfixed in the same solution for 2 h at room temperature and washed in PBS. Segments of stria vascularis and organ of Corti together with Reissner's membrane were dissected out of the inner ear specimens using a fine needle. Samples were permeabilized in 1% Triton X-100 for 30 min and washed with PBS, followed by overnight incubation at 4° C. in blocking solution. The tissues were then stained with rhodamine-phalloidin (1:100 dilution; Molecular Probes, Eugene, Oreg., USA). After washing in PBS, the tissues were mounted using the ProLong Antifade kit (Molecular Probes, Eugene, Oreg., USA) for 20 min at room temperature. Images of tissues were obtained using a laser scanning confocal microscope (Zeiss LSM 510, Germany).
While the present invention has been disclosed by way preferred embodiments, it is not intended to limit the present invention. Any person of ordinary skill in the art may, without departing from the spirit and scope of the present invention, shall be allowed to perform modification and embellishment. Therefore, the scope of protection of the present invention shall be governed by which defined by the claims attached subsequently.

REFERENCE

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Claims

What is claimed is:

1. A method for treating a method for treating an auditory neuropathy spectrum disorder in a subject comprising transferring a transgene via an adeno-associated virus (AAV) vector to the subject; wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.

2. The method of claim 1, wherein the transgene is Pjvk or PCDH15.

3. The method of claim 1, wherein the AAV vector is an AAV vector comprising an Anc80 capsid protein.

4. The method of claim 3, wherein the AAV vector is a synthetic inner ear hair cell targeting adeno-associated virus (AAV) vector, wherein the vector encodes a capsid having at least about 85% sequence identity to Anc80, and comprises a promoter selected from the group consisting of an Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter that directs expression of harmonin-a, harmonin-b, or harmonin-c polypeptide.

5. A construct for delivering a transgene to a subject suffering from an auditory neuropathy spectrum disorder, which comprises an adeno-associated virus (AAV) and the nucleic acids designated a transgene;

wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.

6. The construct of claim 5, wherein the transgene is Pjvk or PCDH15.

7. The construct of claim 5, wherein the AAV vector is an AAV vector comprising an Anc80 capsid protein.

8. The construct of claim 7, wherein the AAV vector is a synthetic inner ear hair cell targeting adeno-associated virus (AAV) vector, wherein the vector encodes a capsid having at least about 85% sequence identity to Anc80, and comprises a promoter selected from the group consisting of an Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter that directs expression of harmonin-a, harmonin-b, or harmonin-c polypeptide.