Gene Therapy for Sensorineural Hearing Loss Wade W. Chien,1,2 Elyssa L. Monzack,1 Devin S. McDougald,1 and Lisa L. Cunningham1

Gene therapy is a promising treatment modality that is being explored for several inherited disorders. Multiple human gene therapy clinical trials are currently ongoing, but few are directed at hearing loss. Hearing loss is one of the most prevalent sensory disabilities in the world, and genetics play an important role in the pathophysiology of hearing loss. Gene therapy offers the possibility of restoring hearing by overcoming the functional deficits created by the underlying genetic mutations. In addition, gene therapy could potentially be used to induce hair cell regeneration by delivering genes that are critical to hair cell differentiation into the cochlea. In this review, we examine the promises and challenges of applying gene therapy to the cochlea. We also summarize recent studies that have applied gene therapy to animal models of hearing loss.

a correction is made to genomic DNA, this is not usually the case. Techniques for making specific changes in the sequences of genomic DNA (called “genome editing”) are beginning to be developed in animal models, and trials using these techniques are underway in humans (Manjunath et al. 2013). In general, the term “gene therapy” usually refers to delivery of cDNA or RNA to a cell or tissue. The concept of gene therapy in humans was first proposed by Friedmann and Roblin in 1972 (Friedmann & Roblin 1972). At that time, very little was known about the human genome. Today, the human genome has been sequenced, and human gene therapy has made significant progress in recent years. For example, gene therapy is already in clinical use in patients with Leber’s congenital amaurosis, which causes earlyonset blindness because of mutations in the gene encoding RPE65. Injection of functional copies of RPE65 cDNA into the subretinal space of these patients results in significant improvement in vision (Cideciyan et al. 2009; Maguire et al. 2009; Simonelli et al. 2010). The inner ear makes a good candidate for gene therapy for several reasons: (1) it is reasonably self-contained anatomically, allowing easy and direct delivery of gene therapy; (2) it is a fluid-filled organ, which allows for widespread diffusion of the delivered gene; and (3) mutations in over 100 genes have been shown to cause hearing loss (Lenz & Avraham 2011), which allows the impact of these genetic mutations on different cell types in the inner ear to be studied by quantitative, structural, and physiological analyses.

Key words: Gene therapy, Hair cell regeneration, Hearing loss, Ototoxicity. (Ear & Hearing 2015;36;1–7)

INTRODUCTION Hearing loss is one of the most common morbidities affecting the U.S. population. Its prevalence has been estimated to be 16.1 to 45.9% (Gates et al. 1990; Cruickshanks et al. 1998; Agrawal et al. 2008). Numerous studies have demonstrated a consistently negative impact of hearing loss on communication (Dubno et al. 1984; Humes et al. 1994; Wiley et al. 1998; Halling & Humes 2000; Gordon-Salant 2005) and quality of life (Mulrow et al. 1990; Carabellese et al. 1993; Dalton et al. 1998; Tsuruoka et al. 2001; Chia et al. 2007). In addition, hearing loss has recently been shown to be a risk factor for dementia (Uhlmann et al. 1989; Ives et al. 1995), and it is associated with decreased functional abilities (Bess et al. 1989; Cacciatore et al. 1999; Strawbridge et al. 2000; Wallhagen et al. 2001; Dalton et al. 2003) and increased mortality (Appollonio et al. 1996; Barnett & Franks 1999). The current therapeutic options for patients with sensorineural hearing loss include hearing amplification and cochlear implantation. While these interventions are effective in many cases, some patients still struggle with the physiologic and psychosocial impacts of deafness. Hearing is mediated by mechanosensory hair cells located within the inner ear. In mammals, mechanosensory hair cells in the cochlea are only generated during a short period of embryonic development (Kelley 2006; White et al. 2006; Oshima et al. 2007; Kelley et al. 2009; Sinkkonen et al. 2011). Thus, if these cells are lost through damage or the natural aging process, they are not replaced, and the result of hair cell loss is permanent sensory impairment. This review is focused on recent advances in the development of gene therapies to prevent or reverse hearing loss. Gene therapy is defined as the delivery of genetic materials (e.g., cDNA, RNAi, etc.) into diseased cells with the hope of exerting therapeutic effects to reverse the underlying disease process. While “gene therapy” may seem to imply that

Gene Therapy Delivery Methods Most gene therapy approaches use viruses to deliver genes to the cells of interest. When selecting the optimal virus for delivering gene therapy, several important factors must be considered. The virus should be specific in delivering functional copies of the gene of interest to the affected cells, and it should be able to do this with high efficiency. Viruses used for gene therapy are modified so that they are replication deficient (Table 1), and in most cases, the viral genes have been replaced with the gene of interest, leaving only the parts of the virus that are necessary for the virus to enter the cells of interest and deliver its cargo (Figs. 1A, B). Therefore, these viruses are often referred to as viral “vectors.” Several types of viral vectors have been used to deliver genetic material to the inner ear in animal studies (Geschwind et al. 1996; Lalwani et al. 1996; Raphael et al. 1996; Dazert et al. 1997; Derby et al. 1999; Han et al. 1999; Holt et al. 1999; Luebke et al. 2001a; Di Pasquale et al. 2005; Kilpatrick et al. 2011; Akil et al. 2012). Adenovirus is a doublestranded DNA virus that infects a wide variety of cell types in humans, and it has been used in several gene therapy clinical trials in humans (Breyer et al. 2001; Jiang et al. 2009). It has a relatively large gene carrying capacity (~7.5–10 kb) and does not require cell division to deliver its genetic cargo (Raphael, et al. 1996; Dazert et al. 1997). This is important because most of the cell types in the mature inner ear are post-mitotic and do

Section on Sensory Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA; and 2Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

1

0196/0202/2015/361-0001/0 • Ear & Hearing • Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved • Printed in the U.S.A. 1

2

CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

TABLE 1.   Glossary Cryptic splice site Cytokine Dominant negative Episome Gene therapy Insertional mutagenesis Replication deficient RNA interference (RNAi) Transduction Viral capsid Viral vector

Mutation which triggers slicing to occur at an abnormal location on the messenger RNA, leading to production of defective gene product A family of signaling molecules secreted by cells A mutation whose gene product adversely affects the normal, wild-type gene product A genetic material that can exist autonomously in the cytoplasm Transfer of genetic materials into a diseased cell to reverse and improve the disease process Disruption of a critical gene in the host genome by viral integration into the host chromosome Lacking the ability to replicate. Viruses can be made replication deficient by removing critical genetic materials necessarily for replication. This makes the viruses incapable of mounting an full-blown infection. The use of RNA molecules to silence a target gene, typically by destruction of mRNAs The process by which genetic material is transferred into a cell by a virus The protein shell of the virus Virus that is used to deliver nucleic acid into cells of interest

not undergo cell division. Adenovirus infects both the hair cells and supporting cells in the inner ear (Kawamoto et al. 2001; Luebke et al. 2001b; Ishimoto et al. 2002). There are at least two disadvantages of using adenovirus for gene therapy in humans. First, adenovirus is immunogenic in humans, and it can elicit a robust inflammatory response, which can cause tissue damage in the host cells (Thaci et al. 2011). Second, the expression of the delivered gene is short-lived because adenovirus does not integrate into the host genome (Raphael et al. 1996; Dazert et al. 1997). For these reasons, the use of adenovirus in human gene therapy clinical trials is limited to disease processes where short-term gene expression is desirable. One of the most commonly used viral vectors in gene therapy is adeno-associated virus (AAV), which is a singlestranded DNA parvovirus. It is nonpathogenic in humans, as it does not cause any known disease. Animal studies indicate that AAV can infect both hair cells and supporting cells in the inner ear (Lalwani et al. 1996, 1998; Kilpatrick et al. 2011). Once it enters the host cell, AAV is thought to either remain as an episome (Table 1) or incorporate into the host genome, resulting in stable, potentially long-term gene expression (Mingozzi

& High 2011). Recombinant AAV vectors with manipulated viral capsids (Table 1) have been created to target infection to specific cell types, and these viral vectors sustain long-term gene expression in liver, lung, muscle, brain, blood vessels, and retina of experimental animals (Rabinowitz & Samulski 2000; Walters et al. 2001). AAV is used as the viral vector for gene therapies to treat Leber’s congenital amaurosis, hemophilia B, α1 antitrypsin deficiency, Duchenne muscular dystrophy, cystic fibrosis, and Parkinson’s disease in humans (Brantly et al. 2006; Manno et al. 2006; Kaplitt et al. 2007; Moss et al. 2007; Maguire et al. 2008, 2009; Mendell et al. 2010; LeWitt et al. 2011). One disadvantage of AAV for gene therapy is that it has a small gene carrying capacity (~4–5 kb); therefore, genes with cDNA more than 5 kb are too large to use this vector for gene therapy. In addition, some studies have shown AAV can elicit an immune response in the host, as neutralizing antibodies have been found after AAV gene therapy administration in nonhuman primates (Jiang et al. 2006; Mingozzi et al. 2007). Other viruses that can infect human cells (and therefore might be useful as gene therapy vectors) include herpes simplex virus (HSV), vaccinia virus, and lentivirus. HSV is a

Fig. 1. Gene therapy delivery using viral vectors. A, Replication-deficient viruses are developed by removing the viral DNA and leaving only the viral capsid. The gene of interest (GOI) is then packaged into the viral capsid, and the virus delivers the GOI into the host cell, where is it transported into the nucleus. The GOI can form an episome in certain viruses or it can integrate into the host genome in other viruses. B, One potential goal of cochlear gene therapy is to deliver corrective copies of a GOI into the mechanosensory hair cells in the cochlea to restore their functions. A color version is available online.



CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

double-stranded DNA virus that can effectively deliver genes to mammalian neurons. It has a very large gene carrying capacity (~10–100 kb), and it can infect post-mitotic cells. In the inner ear, HSV infects spiral ganglion neurons as well as supporting cells (Geschwind et al. 1996; Derby et al. 1999). The disadvantage of using HSV for gene therapy is that it is pathogenic in humans—it can cause herpes stomatitis, genital herpes, Bell’s palsy, and even encephalitis. Vaccinia virus belongs to the poxvirus family, and it has been shown to infect both hair cells and mesenchymal cells in the inner ear (Derby et al. 1999). Unfortunately, it elicits a robust inflammatory response in the cochlea following gene delivery (Derby et al. 1999) and has not been widely studied as a gene delivery vehicle to the inner ear. Lentivirus is a retrovirus that can infect nondividing cells. It integrates into the host genome, resulting in stable, long-term gene expression (Han et al. 1999). Lentivirus infects spiral ganglion cells and the spiral ligament in the mammalian inner ear (Han et al. 1999). However, the fact that it integrates into the host genome raises concerns about the potential for insertional mutagenesis (Table 1), which occurs when the virus integrates into the host genome at a locus that results in disruption of another critical gene. In an effort to circumvent some of the problems associated with viral vectors (such as small carrying capacity, immunogenicity, and insertional mutagenesis), several nonviral gene therapy vectors have also been tested. One such nonviral approach utilizes liposomes, which are composed of a bilayer of lipid molecules that encircles the genetic materials to be delivered. When liposomes come into contact with host cells, the liposomal bilayer integrates with the host plasma membrane, and the therapeutic genetic material is released into the host cells (Leventis & Silvius 1990). Liposomes have theoretically unlimited carrying capacity, and they do not trigger any immune response in the host cells (Leventis & Silvius 1990). However, because the genetic material does not integrate into the host genome, the expression of the gene of interest is transient. In addition, liposome’s lack of cell specificity and low transfection efficiency has limited its usage in gene therapy clinical trials. Liposomes have been shown to deliver genes to hair cells and supporting cells in the mammalian cochlea, but the host cells only express the therapeutic gene for a short period of time (approximately 14 days) after gene delivery (Wareing et al. 1999; Staecker et al. 2001).

Surgical Approaches for Gene Therapy Delivery There are several surgical approaches for delivering gene therapy to the inner ear. Perhaps the least traumatic delivery method is intratympanic injection. In this approach, the gene delivery vehicle (e.g., viral vector or liposomes) containing the gene of interest is injected through the tympanic membrane into the middle ear (Table  2). Because the viral particles or liposomes must then diffuse into the inner ear through the round window membrane, this method results in little inner ear uptake of the gene of interest (Dazert et al. 2001). Another method for gene delivery to the cochlea utilizes a gelatin sponge soaked with viral vector that is applied to the round window membrane (Jero et al. 2001). Unfortunately, the transduction (Table 1) efficiency with this method is also low. For more direct routes of gene delivery to the inner ear, canalostomy, round window membrane injection, cochleostomy, and endolymphatic sac injection have been studied (Yamasoba

3

et al. 1999; Kawamoto et al. 2001; Praetorius et al. 2003; Akil et al. 2012; Okada et al. 2012; Xia et al. 2012). In the canalostomy approach, an opening is made in the posterior semicircular canal, and the viral vectors containing the gene of interest are injected into the inner ear. This approach has been shown in animal studies to cause minimal hearing loss, but the pattern of viral transduction is variable, with some studies reporting transduction of most vestibular cell types and some reporting cochlear transduction (Kawamoto et al. 2001; Praetorius et al. 2003; Okada et al. 2012). In the round window approach, the viral vector containing the gene of interest is injected through the round window membrane into scala tympani. AAV delivered via the round window infects both inner and outer hair cells, supporting cells, and spiral ganglion cells (Akil et al. 2012; Xia et al. 2012). In the cochleostomy approach, an opening is made usually in the basal turn of the cochlea, and the viral vector containing the gene of interest is injected into scala media (Kilpatrick et al. 2011; Akil et al. 2012), though this approach could also be used to access scala tympani. The cochleostomy approach is perhaps the most direct way of delivering genes to endolymph, and AAV delivered via the cochleostomy approach infects all cell types in the cochlea (Kilpatrick et al. 2011). However, cochleostomy is associated with a higher incidence of sensorineural hearing loss compared to the other surgical approaches (Kawamoto et al. 2001; Praetorius et al. 2003). In the endolymphatic sac approach, the viral vector carrying the gene of interest is injected into the endolymphatic sac. Adenovirus delivered via the endolymphatic sac approach infects cells in the endolymphatic sac and duct, vestibular end organs, stria vascularis, Reissner’s membrane, and Hensen’s cells in the cochlea (Yamasoba et al. 1999). Hearing outcome was not reported in this study. Therefore, the round window and cochleostomy approaches appear to result in the most efficient delivery of therapeutic genes to hair cells and supporting cells. One consideration in choosing the cochlear compartment (i.e., endolymphatic versus perilymphatic space) to access is the volume required for effective gene therapy. In mouse, the volume of cochlear endolymph was reported to be 0.19 μl and perilymphatic space (scala tympani and scala vestibule) was 0.6 μl (Thorne et al. 1999). Administration of excessive volume may cause hydraulic trauma to the cochlea. Therefore, when larger volume is required for gene therapy, accessing the perilymphatic space may be preferable because of its larger volume compared to the endolymphatic space.

Gene Therapy for Genetic Hearing Loss Genetic hearing loss is among the most common inherited sensory disorders, affecting 1 in every 1000 births (Morton 1991). Nonsyndromic hearing loss accounts for ~70% of congenital deafness, whereas syndromic hearing loss accounts for the other 30%. More than 130 genetic loci have been linked to nonsyndromic hereditary hearing loss and more than 60 genes have been identified at these loci (Lenz & Avraham 2011). The manifestation of hearing loss is often different in autosomal dominant and recessive inheritance patterns. In autosomal dominant hearing loss, the hearing loss often has a delayed onset and it is progressive, whereas in autosomal recessive hearing loss, the hearing loss tends to be prelingual in onset and is more severe. In autosomal recessive hearing loss, the underlying mutation is usually a loss-of-function mutation, resulting

4

CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

TABLE 2.   Surgical approaches for inner ear gene delivery Approach Intratympanic injection Canalostomy

Round window Cochleostomy

Endolymphatic sac

Space Accessed

Advantages

Disadvantages

References (Animal Used)

Middle ear Posterior canal

Nontraumatic to inner ear Minimal inner ear uptake Dazert et al. 2001 (rat) Causes minimal hearing loss Variable cochlear transduction Kawamoto et al. 2001 (mouse); Okada et al. 2012 (mouse); Praetorius et al., 2003 (mouse) Scala tympani Higher cochlear tranduction May cause small amount of Akil et al. 2012 (mouse); hearing loss Xia et al. 2012 (mouse) Scala media High cochlear tranduction May cause significant hearing Akil et al. 2012 (mouse); loss Kilpatrick et al. 2011 (mouse); Praetorius et al. 2003 (mouse) Endolymphatic sac High transduction of Minimal cochlear Yamasoba et al. 1999 vestibular end organs transduction (guinea pig)

in gene products that have reduced or no function. Therefore, gene therapy should be aimed at replacing the nonfunctional gene products by delivering functional copies of the gene (e.g., cDNA). In autosomal dominant hearing loss, the underlying mutation is usually a gain-of-function or dominant-negative mutation, in which gene products either function abnormally or interfere with normal gene products, respectively. In these cases, gene therapy is aimed at silencing the mutated copy of the gene to restore normal function (e.g., RNA interference or RNAi). Several recent studies (discussed below) offer promising data on gene therapy in animal models of human hereditary hearing loss. VGLUT3  •  Glutamate is the major excitatory neurotransmitter of the organ of Corti. Mice lacking vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapse (Seal et al. 2008). The inner hair cells are morphologically normal in these animals, but there is a decrease in the number of spiral ganglion cells compared to wild-type animals (Seal et al. 2008). Humans that carry a missense mutation in the SLC17A8 gene, which encodes VGLUT3, have autosomal dominant, nonsyndromic hearing loss (DFNA25) (Ruel et al. 2008). In a recent study by Akil et al. (2012), AAV was used to deliver functional copies of the Vglut3 cDNA to the cochleae of Vglut3 knockout mice. In animals less than 12 days of age, delivery of the Vglut3 gene resulted in correction of the hearing loss, as measured by auditory brainstem evoked response (ABR) thresholds within 2 weeks. The normal ABR thresholds were maintained in some animals as long as 1.5 years after gene therapy was delivered. Immunohistochemical and microscopic data indicated that the expression of VGLUT3 was restored in the inner hair cells of these Vglut3 knockout mice. This is the first study in which gene therapy was successfully applied to an animal model of deafness caused by a loss-of-function mutation. GJB2  •  Connexin 26 mutations are among the most common causes of genetic hearing loss in humans (Denoyelle et al. 1997; Kelsell et al. 1997; Estivill et al. 1998; Green et al. 1999). Connexin 26 is encoded by the gap junction protein beta 2 (GJB2) gene. In a recent study by Yu et al., normal copies of the Gjb2 gene were delivered by AAV to the cochlea of Gjb2 knockout mice (Yu et al. 2014). The authors found Gjb2 gene therapy was able to reestablish intercellular gap junction network in the mutant mouse cochlea.

While most mutant alleles of this gene are inherited in an autosomal recessive fashion, some mutant alleles are linked to autosomal dominant hearing loss through a dominant-negative effect (Table 1). For example, the GJB2 gene with the p.R75W mutation (GJB2R75W) impairs normal gap junction formation, leading to hearing loss. In a study by Maeda et al. (2005), Gjb2R75W was delivered to the cochleas of wild-type mice using liposomes (Maeda et al. 2005). Gjb2R75W caused a significant ABR threshold shift (~20 dB) in these animals. The authors then used RNAi to suppress the expression of Gjb2R75W. When Gjb2R75W was administered into the cochlea along with RNAi that specifically silences the Gjb2R75W expression, Gjb2R75W was significantly reduced and the ABR threshold shift caused by Gjb2R75W was avoided. This was the first study in which gene therapy was successfully applied to an animal model of genetic hearing loss caused by a dominant-negative mutation, and it illustrates the feasibility of using gene therapy to deliver RNAi to inhibit expression of a mutated gene. USH1C  •  Usher syndrome is one of the most common syndromic hearing losses. Patients with Usher syndrome present with hearing loss, vestibular dysfunction, and retinitis pigmentosa, which can lead to blindness. Mutations in the USH1C gene, which encodes the protein harmonin, have been shown to cause Usher syndrome. A recent report describes the use of anti-sense oligonucleotides (ASO) as a form of gene therapy to correct the underlying genetic mutation in Ush1c gene in a mouse model of Usher syndrome in which a mutation in the Ush1c gene results in a cryptic splice site (Lentz et al. 2013). The ASO was designed to bind to the pre-mRNA at the mutated site, thereby blocking the cryptic splice site and allowing normal splicing to occur. When ASO was administered intraperitoneally to mice with Ush1c mutation, there was a significant improvement in hearing as well as vestibular function. In addition, ASO administration also prolonged hair cell survival.

Hair Cell Regeneration ATOH1  •  Sensorineural hearing loss most commonly results from degeneration of cochlear hair cells. While hair cells are regenerated in nonmammalian vertebrates (Corwin & Cotanche 1988; Ryals & Rubel 1988; Brignull et al. 2009), the mature mammalian inner ear has an extremely limited capacity for regeneration. The gene ATOH1 encodes a basic helix-loop-helix transcription factor that is critical for hair cell differentiation (Bermingham et al. 1999; Chen et al. 2002). In a study by Izu-



CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

mikawa et al., guinea pigs were first deafened by systemic coadministration of ethacrynic acid and the aminoglycoside antibiotic kanamycin. This treatment led to a complete loss of hair cells within 3 days. Hearing loss in these animals was confirmed by ABR. The Atoh1 gene was then delivered into the deafened cochleae using an adenovirus vector via the cochleostomy approach. The authors showed that some hair cells were restored and there was an improvement in ABR thresholds after Atoh1 gene delivery (Izumikawa et al. 2005). One of the challenges of a study like this is the fact that it is very difficult to control for the variability in hair cell lesions. Therefore, these results have yet to be replicated. Atoh1 gene therapy has also been shown to restore vestibular function in animal models of vestibulopathy induced by ototoxic drugs (Staecker et al. 2007; Schlecker et al. 2011). It is important to note that while Atoh1 plays an important role in hair cell differentiation, it is not the only gene involved in this process, and therefore additional work needs to be done to replicate and expand on these results. Atoh1 gene therapy has also been studied in the setting of noise-induced hearing loss. Noise trauma is thought to induce hearing loss by causing irreversible damage to hair cell stereocilia bundles, leading to hair cell death (Liberman & Kiang 1984). In a study by Yang et al., guinea pigs were exposed to repeated noise stimuli that simulated gunfire (Yang et al. 2012), causing over 60 dB of threshold elevation as measured by ABR. Electron microscopy revealed significant damage to the stereocilia and loss of inner and outer hair cells. When Atoh1 was delivered to the noise-damaged cochleae using adenovirus via the round window approach, some of the stereocilia bundles were restored and many more hair cells survived in the treated cochleae as compared to the untreated ones. The ABR thresholds were also improved by 35 to 40 dB in the Atoh1-treated ears. Taken together, Atoh1 gene therapy may be a promising pathway of inducing hair cell regeneration and providing hair cell protection. miRNA  •  Micro-RNAs (miRNAs) are small, noncoding RNA sequences that regulate gene expression by preventing mRNA translation. This process is referred to as RNAi. miRNAs play important roles in inner ear development and possibly in hair cell regeneration (Rudnicki & Avraham 2012). In a study by Li et al., members of the miRNA 183 family (miR-183, miR96, and miR-182) were overexpressed via injection of synthetic miRNAs into zebrafish embryos (Li et al. 2010). Overexpression of miR-96 and miR-182 resulted in formation of ectopic and supernumerary hair cells. In contrast, knockdown of the miRNA 183 family using complimentary morpholino oligonucleotides resulted in a reduced number of hair cells in the inner ear (Li et al. 2010). Similarly, Frucht et al. reported that overexpression of miRNA 181s (miR181a) can stimulate hair cell proliferation in the chick basilar papilla, as measured by increased BrdU incorporation (Frucht et al. 2010). These studies suggest that gene therapies using miRNAs may be a strategy for inducing hair cell regeneration.

Gene Therapy and Autoimmune Inner Ear Disease Patients with autoimmune inner ear disease produce autoantibodies against various inner ear antigens, leading to bilateral, progressive sensorineural hearing loss (McCabe 1979; Baek et al. 2006). Animal models of autoimmune hearing loss are generated by immunization with β-tubulin, one of the inner

5

ear antigens (Cai et al. 2009; Zhou et al. 2011). In a study by Zhou et al. (2012), autoimmune hearing loss was induced by immunization against β-tubulin in mice that are deficient in interleukin-10 (IL-10) (Zhou et al. 2012). IL-10 is a cytokine (Table 1) that mediates its anti-inflammatory actions by inhibiting the production of proinflammatory cytokines involved in monocyte/macrophage activation (Anderson et al. 2004; Maynard & Weaver 2008). Mice with autoimmune ear disease had more prolonged periods of hearing loss if they were deficient in IL-10 than if they had normal levels of IL-10. IL-10 gene therapy rescued the hearing of these IL-10-deficient mice. This study serves as a proof of concept for using gene therapy as a treatment for autoimmune inner ear disease.

Gene Therapy for Neuronal Preservation Another strategy for using gene therapy to treat sensorineural hearing loss is to deliver genes that can protect against hair cell and/or neuronal loss. Wise et al. (2011) used adenovirus to deliver cDNAs encoding brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) to scala media in guinea pigs that had been deafened by furosemide and kanamycin administration (Wise et al. 2011). BDNF and NT3 play important roles in the development and maintenance of spiral ganglion neurons (Fritzsch et al. 1997; Altschuler et al. 1999). The cochleas that were treated with BDNF and NT3 gene therapy had higher spiral ganglion neuron survival than cochleas that did not receive gene therapy (Wise et al. 2011). In another study, Leake et al. delivered recombinant BDNF using an osmotic pump into cat cochleas (Leake et al. 2013). These cats were deafened previously by neomycin administration and underwent cochlear implantation, which electrically stimulates the ears. The authors found that BDNF administration with cochlear implant electrical stimulation significantly increased the spiral ganglion density. These data indicate that gene therapy with factors that promote cell survival can be used to prevent death of specific cell types in the cochlea, and they suggest that protective gene therapies may hold promise to prevent death of cells in the inner ear.

CONCLUSIONS Gene therapy offers the possibility of treating sensorineural hearing loss by restoring and/or preserving the functions of cells in the inner ear. Numerous strategies can be developed for inner ear gene therapy to treat sensorineural hearing loss, including correcting genetic mutations, preserving and preventing loss of specific cell types (hair cells, spiral ganglion neurons, supporting cells, etc.), and possibly eventually inducing hair cell regeneration. Promising data from recent studies have advanced the development of cochlear gene therapies toward eventual use in humans.

ACKNOWLEDGMENTS We thank Dr. Andrew Griffith, Dr. Carmen Brewer, Dr. Thomas Friedman, and Dr. Tracy Fitzgerald for their review of the article. The authors have no conflict of interest to declare. Address for correspondence: Wade W. Chien, MD, Johns Hopkins School of Medicine, 601 N. Caroline Street, 6th Floor, Baltimore, MD 21287, USA. E-mail: [email protected] Received March 31, 2014; accepted June 18, 2014.

6

CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

REFERENCES Agrawal, Y., Platz, E. A., Niparko, J. K. (2008). Prevalence of hearing loss and differences by demographic characteristics among US adults: data from the National Health and Nutrition Examination Survey, 1999-2004. Arch Intern Med, 168, 1522–1530. Akil, O., Seal, R. P., Burke, K., et al. (2012). Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron, 75, 283–293. Altschuler, R. A., Cho, Y., Ylikoski, J., et al. (1999). Rescue and regrowth of sensory nerves following deafferentation by neurotrophic factors. Ann N Y Acad Sci, 884, 305–311. Anderson, A. C., Reddy, J., Nazareno, R., et al. (2004). IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice. J Immunol, 173, 828–834. Appollonio, I., Carabellese, C., Frattola, L., et al. (1996). Effects of sensory aids on the quality of life and mortality of elderly people: A multivariate analysis. Age Ageing, 25, 89–96. Baek, M. J., Park, H. M., Johnson, J. M., et al. (2006). Increased frequencies of cochlin-specific T cells in patients with autoimmune sensorineural hearing loss. J Immunol, 177, 4203–4210. Barnett, S., & Franks, P. (1999). Deafness and mortality: analyses of linked data from the National Health Interview Survey and National Death Index. Public Health Rep, 114, 330–336. Bermingham, N. A., Hassan, B. A., Price, S. D., et al. (1999). Math1: An essential gene for the generation of inner ear hair cells. Science, 284, 1837–1841. Bess, F. H., Lichtenstein, M. J., Logan, S. A., et al. (1989). Hearing impairment as a determinant of function in the elderly. J Am Geriatr Soc, 37, 123–128. Brantly, M. L., Spencer, L. T., Humphries, M., et al. (2006). Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults. Hum Gene Ther, 17, 1177–1186. Breyer, B., Jiang, W., Cheng, H., et al. (2001). Adenoviral vector-mediated gene transfer for human gene therapy. Curr Gene Ther, 1, 149–162. Brignull, H. R., Raible, D. W., Stone, J. S. (2009). Feathers and fins: Nonmammalian models for hair cell regeneration. Brain Res, 1277, 12–23. Cacciatore, F., Napoli, C., Abete, P., et al. (1999). Quality of life determinants and hearing function in an elderly population: Osservatorio Geriatrico Campano Study Group. Gerontology, 45, 323–328. Cai, Q., Du, X., Zhou, B., et al. (2009). Induction of tolerance by oral administration of beta-tubulin in an animal model of autoimmune inner ear disease. ORL J Otorhinolaryngol Relat Spec, 71, 135–141. Carabellese, C., Appollonio, I., Rozzini, R., et al. (1993). Sensory impairment and quality of life in a community elderly population. J Am Geriatr Soc, 41, 401–407. Chen, P., Johnson, J. E., Zoghbi, H. Y., et al. (2002). The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development, 129, 2495–2505. Chia, E. M., Wang, J. J., Rochtchina, E., et al. (2007). Hearing impairment and health-related quality of life: The Blue Mountains Hearing Study. Ear Hear, 28, 187–195. Cideciyan, A. V., Hauswirth, W. W., Aleman, T. S., et al. (2009). Vision 1 year after gene therapy for Leber’s congenital amaurosis. N Engl J Med, 361, 725–727. Corwin, J. T., & Cotanche, D. A. (1988). Regeneration of sensory hair cells after acoustic trauma. Science, 240, 1772–1774. Cruickshanks, K. J., Wiley, T. L., Tweed, T. S., et al. (1998). Prevalence of hearing loss in older adults in Beaver Dam, Wisconsin. The Epidemiology of Hearing Loss Study. Am J Epidemiol, 148, 879–886. Dalton, D. S., Cruickshanks, K. J., Klein, B. E., et al. (2003). The impact of hearing loss on quality of life in older adults. Gerontologist, 43, 661–668. Dalton, D. S., Cruickshanks, K. J., Klein, R., et al. (1998). Association of NIDDM and hearing loss. Diabetes Care, 21, 1540–1544. Dazert, S., Aletsee, C., Brors, D., et al. (2001). In vivo adenoviral transduction of the neonatal rat cochlea and middle ear. Hear Res, 151, 30–40. Dazert, S., Battaglia, A., Ryan, A. F. (1997). Transfection of neonatal rat cochlear cells in vitro with an adenovirus vector. Int J Dev Neurosci, 15, 595–600. Denoyelle, F., Weil, D., Maw, M. A., et al. (1997). Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. Hum Mol Genet, 6, 2173–2177. Derby, M. L., Sena-Esteves, M., Breakefield, X. O., et al. (1999). Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors. Hear Res, 134, 1–8.

Di Pasquale, G., Rzadzinska, A., Schneider, M. E., et al. (2005). A novel bovine virus efficiently transduces inner ear neuroepithelial cells. Mol Ther, 11, 849–855. Dubno, J. R., Dirks, D. D., Morgan, D. E. (1984). Effects of age and mild hearing loss on speech recognition in noise. J Acoust Soc Am, 76, 87–96. Estivill, X., Fortina, P., Surrey, S., et al. (1998). Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet, 351, 394–398. Friedmann, T., & Roblin, R. (1972). Gene therapy for human genetic disease? Science, 175, 949–955. Fritzsch, B., Silos-Santiago, I., Bianchi, L. M., et al. (1997). The role of neurotrophic factors in regulating the development of inner ear innervation. Trends Neurosci, 20, 159–164. Frucht, C. S., Uduman, M., Duke, J. L., et al. (2010). Gene expression analysis of forskolin treated basilar papillae identifies microRNA181a as a mediator of proliferation. PLoS One, 5, e11502. Gates, G. A., Cooper, J. C., Jr., Kannel, W. B., et al. (1990). Hearing in the elderly: The Framingham cohort, 1983–1985. Part I. Basic audiometric test results. Ear Hear, 11(4), 247–256. Geschwind, M. D., Hartnick, C. J., Liu, W., et al. (1996). Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. Hum Gene Ther, 7, 173–182. Gordon-Salant, S. (2005). Hearing loss and aging: new research findings and clinical implications. J Rehabil Res Dev, 42(4 Suppl 2), 9–24. Green, G. E., Scott, D. A., McDonald, J. M., et al. (1999). Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness. JAMA, 281, 2211–2216. Halling, D. C., & Humes, L. E. (2000). Factors affecting the recognition of reverberant speech by elderly listeners. J Speech Lang Hear Res, 43, 414–431. Han, J. J., Mhatre, A. N., Wareing, M., et al. (1999). Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector. Hum Gene Ther, 10, 1867–1873. Holt, J. R., Johns, D. C., Wang, S., et al. (1999). Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. J Neurophysiol, 81, 1881–1888. Humes, L. E., Watson, B. U., Christensen, L. A., et al. (1994). Factors associated with individual differences in clinical measures of speech recognition among the elderly. J Speech Hear Res, 37, 465–474. Ishimoto, S., Kawamoto, K., Kanzaki, S., et al. (2002). Gene transfer into supporting cells of the organ of Corti. Hear Res, 173, 187–197. Ives, D. G., Bonino, P., Traven, N. D., et al. (1995). Characteristics and comorbidities of rural older adults with hearing impairment. J Am Geriatr Soc, 43, 803–806. Izumikawa, M., Minoda, R., Kawamoto, K., et al. (2005). Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med, 11, 271–276. Jero, J., Mhatre, A. N., Tseng, C. J., et al. (2001). Cochlear gene delivery through an intact round window membrane in mouse. Hum Gene Ther, 12, 539–548. Jiang, H., Couto, L. B., Patarroyo-White, S., et al. (2006). Effects of transient immunosuppression on adenoassociated, virus-mediated, liverdirected gene transfer in rhesus macaques and implications for human gene therapy. Blood, 108, 3321–3328. Jiang, H., Gomez-Manzano, C., Lang, F. F., et al. (2009). Oncolytic adenovirus: preclinical and clinical studies in patients with human malignant gliomas. Curr Gene Ther, 9, 422–427. Kaplitt, M. G., Feigin, A., Tang, C., et al. (2007). Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: An open label, phase I trial. Lancet, 369, 2097–2105. Kawamoto, K., Oh, S. H., Kanzaki, S., et al. (2001). The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice. Mol Ther, 4, 575–585. Kelley, M. W. (2006). Regulation of cell fate in the sensory epithelia of the inner ear. Nat Rev Neurosci, 7, 837–849. Kelley, M. W., Driver, E. C., Puligilla, C. (2009). Regulation of cell fate and patterning in the developing mammalian cochlea. Curr Opin Otolaryngol Head Neck Surg, 17, 381–387. Kelsell, D. P., Dunlop, J., Stevens, H. P., et al. (1997). Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature, 387, 80–83. Kilpatrick, L. A., Li, Q., Yang, J., et al. (2011). Adeno-associated virusmediated gene delivery into the scala media of the normal and deafened adult mouse ear. Gene Ther, 18, 569–578. Lalwani, A., Walsh, B., Reilly, P., et al. (1998). Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus. Gene Ther, 5, 277–281.



CHIEN ET AL. / EAR & HEARING, VOL. 36, NO. 1, 1–7

Lalwani, A. K., Walsh, B. J., Reilly, P. G., et al. (1996). Development of in vivo gene therapy for hearing disorders: Introduction of adeno-associated virus into the cochlea of the guinea pig. Gene Ther, 3, 588–592. Leake, P. A., Stakhovskaya, O., Hetherington, A., et al. (2013). Effects of brain-derived neurotrophic factor (BDNF) and electrical stimulation on survival and function of cochlear spiral ganglion neurons in deafened, developing cats. J Assoc Res Otolaryngol, 14, 187–211. Lentz, J. J., Jodelka, F. M., Hinrich, A. J., et al. (2013). Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness. Nat Med, 19, 345–350. Lenz, D. R., & Avraham, K. B. (2011). Hereditary hearing loss: From human mutation to mechanism. Hear Res, 281, 3–10. Leventis, R., & Silvius, J. R. (1990). Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim Biophys Acta, 1023, 124–132. LeWitt, P. A., Rezai, A. R., Leehey, M. A., et al. (2011). AAV2-GAD gene therapy for advanced Parkinson’s disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol, 10, 309–319. Li, H., Kloosterman, W., Fekete, D. M. (2010). MicroRNA-183 family members regulate sensorineural fates in the inner ear. J Neurosci, 30, 3254–3263. Liberman, M. C., & Kiang, N. Y. (1984). Single-neuron labeling and chronic cochlear pathology. IV. Stereocilia damage and alterations in rate- and phase-level functions. Hear Res, 16, 75–90. Luebke, A. E., Foster, P. K., Muller, C. D., et al. (2001). Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer. Hum Gene Ther, 12, 773–781. Luebke, A. E., Steiger, J. D., Hodges, B. L., et al. (2001). A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function. Gene Ther, 8, 789–794. Maeda, Y., Fukushima, K., Nishizaki, K., et al. (2005). In vitro and in vivo suppression of GJB2 expression by RNA interference. Hum Mol Genet, 14, 1641–1650. Maguire, A. M., High, K. A., Auricchio, A., et al. (2009). Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: A phase 1 dose-escalation trial. Lancet, 374, 1597–1605. Maguire, A. M., Simonelli, F., Pierce, E. A., et al. (2008). Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med, 358, 2240–2248. Manjunath, N., Yi, G., Dang, Y., et al. (2013). Newer gene editing technologies toward HIV gene therapy. Viruses, 5, 2748–2766. Manno, C. S., Pierce, G. F., Arruda, V. R., et al. (2006). Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 12, 342–347. Maynard, C. L., & Weaver, C. T. (2008). Diversity in the contribution of interleukin-10 to T-cell-mediated immune regulation. Immunol Rev, 226, 219–233. McCabe, B. F. (1979). Autoimmune sensorineural hearing loss. Ann Otol Rhinol Laryngol, 88(5 Pt 1), 585–589. Mendell, J. R., Campbell, K., Rodino-Klapac, L., et al. (2010). Dystrophin immunity in Duchenne’s muscular dystrophy. N Engl J Med, 363, 1429–1437. Mingozzi, F., Hasbrouck, N. C., Basner-Tschakarjan, E., et al. (2007). Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver. Blood, 110, 2334–2341. Mingozzi, F., & High, K. A. (2011). Therapeutic in vivo gene transfer for genetic disease using AAV: Progress and challenges. Nat Rev Genet, 12, 341–355. Morton, N. E. (1991). Genetic epidemiology of hearing impairment. Ann N Y Acad Sci, 630, 16–31. Moss, R. B., Milla, C., Colombo, J., et al. (2007). Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: A randomized placebo-controlled phase 2B trial. Hum Gene Ther, 18, 726–732. Mulrow, C. D., Aguilar, C., Endicott, J. E., et al. (1990). Quality-of-life changes and hearing impairment. A randomized trial. Ann Intern Med, 113, 188–194. Okada, H., Iizuka, T., Mochizuki, H., et al. (2012). Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors. Otol Neurotol, 33, 655–659. Oshima, K., Grimm, C. M., Corrales, C. E., et al. (2007). Differential distribution of stem cells in the auditory and vestibular organs of the inner ear. J Assoc Res Otolaryngol, 8, 18–31. Praetorius, M., Baker, K., Weich, C. M., et al. (2003). Hearing preservation after inner ear gene therapy: The effect of vector and surgical approach. ORL J Otorhinolaryngol Relat Spec, 65, 211–214. Rabinowitz, J. E., & Samulski, R. J. (2000). Building a better vector: the manipulation of AAV virions. Virology, 278, 301–308.

7

Raphael, Y., Frisancho, J. C., Roessler, B. J. (1996). Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo. Neurosci Lett, 207, 137–141. Rudnicki, A., & Avraham, K. B. (2012). microRNAs: the art of silencing in the ear. EMBO Mol Med, 4, 849–859. Ruel, J., Emery, S., Nouvian, R., et al. (2008). Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice. Am J Hum Genet, 83, 278–292. Ryals, B. M., & Rubel, E. W. (1988). Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science, 240, 1774–1776. Schlecker, C., Praetorius, M., Brough, D. E., et al. (2011). Selective atonal gene delivery improves balance function in a mouse model of vestibular disease. Gene Ther, 18, 884–890. Seal, R. P., Akil, O., Yi, E., et al. (2008). Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron, 57, 263–275. Simonelli, F., Maguire, A. M., Testa, F., et al. (2010). Gene therapy for Leber’s congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther, 18, 643–650. Sinkkonen, S. T., Chai, R., Jan, T. A., et al. (2011). Intrinsic regenerative potential of murine cochlear supporting cells. Sci Rep, 1, 26. Staecker, H., Li, D., O’Malley, B. W. Jr, et al. (2001). Gene expression in the mammalian cochlea: A study of multiple vector systems. Acta Otolaryngol, 121, 157–163. Staecker, H., Praetorius, M., Baker, K., et al. (2007). Vestibular hair cell regeneration and restoration of balance function induced by math1 gene transfer. Otol Neurotol, 28, 223–231. Strawbridge, W. J., Wallhagen, M. I., Shema, S. J., et al. (2000). Negative consequences of hearing impairment in old age: A longitudinal analysis. Gerontologist, 40, 320–326. Thaci, B., Ulasov, I. V., Wainwright, D. A., et al. (2011). The challenge for gene therapy: Innate immune response to adenoviruses. Oncotarget, 2, 113–121. Thorne, M., Salt, A. N., DeMott, J. E., et al. (1999). Cochlear fluid space dimensions for six species derived from reconstructions of three-dimensional magnetic resonance images. Laryngoscope, 109, 1661–1668. Tsuruoka, H., Masuda, S., Ukai, K., et al. (2001). Hearing impairment and quality of life for the elderly in nursing homes. Auris Nasus Larynx, 28, 45–54. Uhlmann, R. F., Larson, E. B., Rees, T. S., et al. (1989). Relationship of hearing impairment to dementia and cognitive dysfunction in older adults. JAMA, 261, 1916–1919. Wallhagen, M. I., Strawbridge, W. J., Shema, S. J., et al. (2001). Comparative impact of hearing and vision impairment on subsequent functioning. J Am Geriatr Soc, 49, 1086–1092. Walters, R. W., Yi, S. M., Keshavjee, S., et al. (2001). Binding of adenoassociated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem, 276, 20610–20616. Wareing, M., Mhatre, A. N., Pettis, R., et al. (1999). Cationic liposome mediated transgene expression in the guinea pig cochlea. Hear Res, 128, 61–69. White, P. M., Doetzlhofer, A., Lee, Y. S., et al. (2006). Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature, 441, 984–987. Wiley, T. L., Cruickshanks, K. J., Nondahl, D. M., et al. (1998). Aging and word recognition in competing message. J Am Acad Audiol, 9, 191–198. Wise, A. K., Tu, T., Atkinson, P. J., et al. (2011). The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection. Hear Res, 278, 69–76. Xia, L., Yin, S., Wang, J. (2012). Inner ear gene transfection in neonatal mice using adeno-associated viral vector: A comparison of two approaches. PLoS One, 7, e43218. Yamasoba, T., Yagi, M., Roessler, B. J., et al. (1999). Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac. Hum Gene Ther, 10, 769–774. Yang, S. M., Chen, W., Guo, W. W., et al. (2012). Regeneration of stereocilia of hair cells by forced Atoh1 expression in the adult mammalian cochlea. PLoS One, 7, e46355. Yu, Q., Wang, Y., Chang, Q., et al. (2014). Virally expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice. Gene Ther, 21, 71–80. Zhou, B., Kermany, M. H., Cai, Q., et al. (2012). Experimental autoimmune hearing loss is exacerbated in IL-10-deficient mice and reversed by IL-10 gene transfer. Gene Ther, 19, 228–235. Zhou, B., Kermany, M. H., Glickstein, J., et al. (2011). Murine autoimmune hearing loss mediated by CD4+ T cells specific for beta-tubulin. Clin Immunol, 138(2), 222–230.