Treatment of Peripheral Sensorineural Hearing Loss: Gene Therapy

M Duan1,2,3,4, F Venail5, N Spencer1 and M Mezzina6 1Department of Clinical Neuroscience and Center for Hearing and Communication Research, Building MI-ENT, Karolinska Hospital, Stockholm, Sweden; 2Department of Otolaryngology, Karolinska Hospital, Stockholm, Sweden; 3Department of Clinical Neuroscience, Karolinska Hospital, Stockholm, Sweden; 4Department of Otolaryngology, Anhui Medical University, Hefei, China; 5ENT Department, CHU Gui de Chauliac, Montpellier, France; and 6Genethon – CNRS-UMR 8115, 1bis Rue de l’International, Evry Cedex, France

Noise, chemicals and genetic defects are all common causes ment, discusses the inner ear model in detail and reviews the of irreversible hearing loss, which at present have no cure. efforts that have been made in inner ear gene therapy. Gene therapy may soon be utilized in both the protection and Finally, the proposed next steps will be discussed. The viral the treatment of these exogenous and endogenous sources mediated delivery of neurotrophins and antoxidants offers of hearing loss. Gene therapy technology is rapidly develop- imminent promise in preventing and treating exogenous ing and the inner ear is a particularly feasible model for gene hearing loss and improving cochlear implant therapy. therapy. This review outlines our current understanding of Gene Therapy (2004) 11, S51–S56. doi:10.1038/sj.gt.3302369 the mechanisms behind deafness and prospects for treat-

Keywords: inner ear; spiral ganglion cell; hair cell; adenovirus; adeno-associated virus; lentivirus; neurotrophin; antioxidant; stem cell

Introduction Secondly, various physiological measurement tools have been developed to monitor the function of specific The cochlea contains two types of sensory cells, inner cells (Table 1), and thus assess the efficacy and safety of hair cells (IHC) and outer hair cells (OHC), which carry gene therapy. Cochlear Microphonics (CM) and Oto- out the transduction of sounds to electrical signals. These acoustic emission (OAE) measurements are useful in signals are transmitted to the auditory pathway of the assessing the state of damage of OHCs. Compound brain via spiral ganglion neurons (SGCs). Unfortunately, Action Potentials (CAPs) are recorded to check IHC neither of these sensory or primary neural cells have the function. Similarly, single unit recordings are used in capacity to regenerate. Thus, any injury to the cochlea SGC assessment. More general methods for monitoring can result in an irreversible hearing loss. Despite recent cochlear physiology exist as well. Detection of Endo- developments in medicine, there are still no clinically cochlear Potentials (EPs) helps to determine the presence useful means for curing hearing disorders and protecting of endolymphatic ion balances. A general diagnostics tool auditory function. It is of paramount importance to is Auditory Brainstem Response (ABR) measurements. develop novel and effective methods of treating both Thirdly, the cochlea is unique among sensorineural inherited and acquired hearing loss. Gene therapy systems in that so many genes have been recently cloned technology has improved in recent years, making it a in the mouse and human. Over 90 different genes have promising technique for treating such inner ear dis- now been identified that affect inner ear development orders. The inner ear holds several unique advantages as or function, as well as many loci known to be involved a model for gene therapy. in deafness. Between 1996 and 2000, 19 genes involved Firstly, the cochlea is anatomically well suited for in in nonsyndromic deafness were identified in a larger vivo gene therapy. The relative isolation of the cochlear number of genes implicated in syndromic deafness.1 In compartments minimizes unwanted effects of the intro- addition, a transgenic technique has been demonstrated duced gene into other tissues. The inner ear is fluid- in shaker-2 mice to correct deafness.2 filled, allowing all functionally important cells to be Finally, there are many possibilities for using gene accessed by a transfection reagent. The concentration and therapy in the cochlea to treat deafness. Neurotrophin dosage of complexes introduced to the cochlea can easily gene therapy is an important example. Neurotrophic be modulated with a single injection or longer infusion factors are essential in the development of the inner ear by an osmotic pump. Cochlear endolymph and peri- and in the protection of the adult inner ear sensory cells lymph volumes have been characterized in the guinea against ototoxic chemical and noise induced damage.3 pig, rat and mouse, so adverse effects of high volume Neurotrophin gene therapy is promising both in the and pressure can be avoided. protection against exogenous damage and the regenera- tion after endgenous and exogenous damage. Neuro- Correspondence: Dr M Duan, Center for Hearing and Communication trophins can be applied through in vivo inoculation of Research, Building MI-ENT, Karolinska Hospital, SE-171 76 Stockholm, vectors carrying their genes or more indirectly through Sweden gene therapy-modified stem cells. Treatment of peripheral sensorineural hearing loss M Duan et al S52 Table 1 Methods for observing various cochlear characteristics cisplatin10 and aminoglycoside otoxicity.11 Antioxidants post gene therapy inoculation have been shown to protect the cochlea from the damage caused by these factors.9–11 Further evidence exists in Function Outer hair cell – cochlear otoacoustic emission Outer hair cell – cochlear microphonics particular for superoxide’s role in hearing loss. Super- Inner hair cell – compound action potential oxide dismutase (Sod) knockout mice have an enhanced Spiral ganglion cell – Single unit recording susceptibility to noise trauma, resulting in severe Cochlear fluid – endocochlear potential NIHL.12 Also, the overexpression of this enzyme protects Auditory nerve – auditory brainstem response (ABR) mice from aminoglycoside ototoxicity in the transgenic model.13 Structure Light microscopy Immunocytochemistry Confocal microscopy Role of neurotrophic factors in protecting the inner ear Neurotrophic factors are important in inner ear devel- UltrastructureTransmission electron microscopy opment and adult inner ear protection. Neurotrophin Scanning electron microscopy secretion is reciprocal among hair cells and SGCs. The production of neurotrophin-3 (NT-3) is crucial to the survival of developing type 1 SGCs innervating IHCs, whereas brain-derived neurotrophic factor (BDNF) is Exogenous and endogenous causes required for the survival of type 2 SGCs innervating OHCs and vestibular hair cells.14 Aminoglycoside- of deafness induced degeneration was prevented by the infusion Many antibiotic and anticancer drugs are ototoxic. of NT-3 in 90% of adult SGCs.15,16 Neurotrophins NT-3, Aminoglycoside antibiotics have been successfully used glial-derived neurotrophic factor (GDNF) and BDNF are in the treatment of various infectious diseases for more important in protection of the cochlea against NIHL.4 than 40 years. These drugs are in use today, despite of Finally, vascular endothelial growth factor (VEGF) is also their side effects. Clinical aminoglycoside use can cause important in axonal outgrowth and cochlear sensory severe degeneration of SGCs and hair cells.4 and supporting cell survival.17 Neurotrophin therapy is The anticancer drug cisplatin was found to have promising in the prevention of exogenous hearing loss. ototoxic side effects early on in its clinical trials.5 Hearing The treatment of peripheral hearing disorders with loss induced by cisplatin is typically strongest at high neurotrophic factors had not been widely investigated frequencies (3–8 kHz) in humans, and repeated admin- until very recently. Miller et al18 demonstrated the istrations affect successively lower frequencies accom- enhancement of SGC survival after the kanamycin panied by progressive hearing loss.6 Cisplatin primarily and ethacrynic acid-induced loss of IHCs, through the damages OHCs and auditory nerve fibres.7 chronic infusion of BDNF into the cochlea. Recent results Noise-induced hearing loss (NIHL) is responsible for showed that the infusion of NT-3 not only protects SGCs one-third of acquired sensorineural hearing loss cases. from aminoglycoside ototoxicity but also facilitates NIHL can be temporary or permanent. Those who suffer neurite regrowth.15,16 Moreover, combined BDNF and from NIHL also tend to have increased vulnerability to ciliary neurotrophic factor (CNTF) therapy increases chemical ototoxic insults.8 the survival of SGCs when applied as late as 2 weeks Approximately one in 800 children are born with a following kanamycin treatment.19 This study also used severe to profound genetic-related hearing loss (GRHL). electrical auditory brainstem response measurements to Single-gene defects likely account for the majority of show an enhancement of auditory performance due to childhood deafness cases.1 The most common cause of neurotrophin treatment of the kanamyicn insult. This GRHL is the GJB2 (Cx26) mutation. is important because the success of cochlear implant Owing to the lack of proliferation in postnatal inner treatment depends on the presence of SGCs at the site ear sensory epithelia, these problems do not have a cure. of the implant. Cochlear implant therapy could be Gene therapy is the most promising solution for curing improved by applying neurotrophins to the human them. cochlea.

Mechanisms of peripheral hearing loss Monitoring auditory function after gene and prospects for treatment therapy Gene delivery systems must not be cytotoxic, and cells The underlying mechanisms of sensorineural hearing must be able to return to a normal physiological state loss are not completely understood. We do, however, after treatment. Many techniques exist to assess cochlear have working hypothesies for surmounting these chal- physiology. First, ABR responses to sound input are used lenges through gene therapy. The following is an outline to noninvasively record activity in all the parts of the of likely mechanisms and how they can be prevented auditory pathway, allowing for a long-term survey. and treated. Using an electrode placed on the round window, the CAP method yields more sensitive results than the ABR. Reactive oxygen species (ROS) and antioxidants Various OAE measurements noninvasively assess the Signiﬁcant ROS generated in the reduction of oxygen to properties of cochlear sound ampliﬁcation related to water include the superoxide anion, hydrogen peroxide OHC function. All of these techniques can be used to and the hydroxyl radical. These ROS are involved in the evaluate the recovery of auditory function after an mechanisms of hearing loss caused by noise trauma,9 invasive surgery or after gene delivery. Additionally,

Gene Therapy Treatment of peripheral sensorineural hearing loss M Duan et al S53 an evaluation of the Stria Vascularis can be performed through the study of EPs. The use of EPs in assessing early anomalies in the scala media could be of particular use in future Meniere’s Disease animal models.

Introducing gene therapy vectors into the cochlea The cochlea, encased by bone, is divided into three fluid compartments called scalae. The scala vestibuli and scala tympani (Figure 1) contain a high-sodium, low- potassium perilymph. The round window is situated at the end of the basal turns of these scalae. The scala media contains endolymph high in potassium and low in sodium. The cochlear sensory apparatus, the organ of Corti, is situated in this compartment. Figure 2 Locating and perfusing the guinea pig round window. Two common access points used for the perfusion or injection of vectors into the perilymph are the round window (RW) and the cochlear bone. The RW is accessible via the tympanic bulla of the rodent middle transfected nearly all tissue types of mice and guinea pig ear (Figure 2). A cochleostomy, a small hole in the cochleae in vivo.22–24 cochlear bone, can be made for access to the scala Evidence has not shown clinically useful transfection tympani. Both of these methods have been shown to efficiencies. A broader investigation of vectors and deliver drugs effectively without hindering rodent 15,16,20 promoters, and the incorporation of nuclear localization cochlear function. The cochleostomy is advanta- sequences and targeting ligands could result in an geous in gene transfer efficacy, while the RW method is efficient vector. Better insight into cell membrane less invasive. Low perfusion volumes should be used in composition and nuclear transport may also result in the cochleostomy. Recently, the placement of gelfoam- improvements. Other methods such as electroporation25 soaked therapeutics on the RW was shown useful in the 26 21 and the gene gun have yielded significant in vitro delivery of certain vectors. The vestibular superior results but have not been developed for effective use semicircular canal can also be accessed via a canalost- in vivo. omy. A cochleostomy can also be made for access to the Another concept is the nonviral vector with virally scala media. derived fusion activity. The hemaglutinating virus of Japan envelope vector recently transfected more than 70 percent of SGCs without affecting ABR performances, Gene therapy in the inner ear after injection to the cisterna magna.27 The injection method raises issues such as dissemination beyond the Nonviral cochlea,28 but this vector may prove more efficient than Liposomes are the traditional nonviral vector used in other nonviral vectors and safer than viral vectors. This inner ear research. They are easy to prepare, can be study addressed not only the prevention but also the complexed with DNA of any size, and have a very low treatment of kanamycin ototoxicity with the incorpora- risk of insertional mutagenesis. Liposome complexed tion of the gene for hepatocyte growth factor. with LacZ and GFP reporter genes have successfully Adenoviral The majority of inner ear gene therapy research has been carried out with replication-deficient (E1À,E3À) Ade- noviral (Ad) vectors, which can be generated at high concentrations29and can accommodate large (8 kb) frag- ments of DNA. They are efficient in infecting many inner ear cell types in vivo, including SGCs.4,17 The replication defective (E1À E3À, polÀ) Ad vector transfects hair cells in vivo.30 It is important to note a large degree of variation in the specific expression among studies. This variation may result from variation in Ad concentrations, Ad vector generations, and methods of vector inoculation and transgenic protein detection. The mechanisms involved in Ad transfection need to be elucidated, ideally through a study of the expression of Ad receptors in the inner ear. Recent studies have used the Ad vector to prevent deafness in animals. Ad-GDNF delivery protects SGCs31 Figure 1 Cochlear architecture, highlighting the sensory hair cells of the from gentamicin ototoxicity. It has also been shown to organ of Corti and the afferent spiral ganglion neurons. protect OHCs from gentamicin ototoxicity,30 in a study

Gene Therapy Treatment of peripheral sensorineural hearing loss M Duan et al S54 where Ad infection of hair cells was not shown. The accessible by the fluid. Less accessible cells such as the reason for the protective effect is unknown, but may spiral ganglia and glial cells have been transfected in be attributed to the secretion of proteins. Combined vitro.42 Based on these early studies, the potential uses for cytokine transforming growth factor (TGF)-b1/GDNF this vector involve the secretion of growth factors and Ad gene therapy improves this protection, but with antibiotics into the perilymph. The possibility of inser- accompanying fibrosis.32 Ad-GDNF delivery also pro- tional mutagenesis must be carefully studied in the inner tects IHCs after an acute cessation of blood supply.33 ear before seriously considering this vector for clinical Antioxidant gene therapy has also proven useful in use. The insertion of genes into the chromosome is also mouse models.34 Neurotrophin and antioxidant gene advantageous, however, in the potential treatment of therapy hold promise in treating deafness.4 genetic hearing loss. For this reason, LV gene therapy The major drawback is the capsid-induced immune holds promise in treating both endogenous and exogen- response,35 typically causing the clearance of infected ous causes of deafness. cells within 10 days. Cytotoxicity has been found in many inner ear studies, likely a consequence of this immune response. Preliminary attempts to address these The Future immune responses have been carried out with two types of immunosuppressants: T-lymphocyte costimulator in- Treating exogenous deafness through viral-mediated hibitors and the anti-inflammatory steroids. Both types expression of secreted products of suppressants can inhibit the Ad-immune response Ad, AAV and LV vectors effectively transfer genes into and allow the production of the second Ad-delivered the inner ear in vivo. AAV and LV vectors have shown 36 transgene. Care must be taken, however, to avoid lengthy transgene expression times and low toxicity. infection in human patients during immunosuppressant Methods have been developed for the inoculation of therapy. vectors into the cochlea as well as the detection of gene products and the assessment of cochlear function. It is Adeno-associated viral particularly promising to influse AAV and LV vectors 37 Adeno-associated viral (AAV) vectors elicit a much less carrying the neurotrophin and antioxidant enzyme genes potent immune response than Ad vectors. In fact, AAVs NT-3, BDNF, GDNF, VEGF and Sod to the guinea pig and have been found to mediate gene expression for up to mouse cochleae in treating chemical and noise-induced 38 6 months, albeit at decreased levels. AAV vectors can inner ear disorders. accommodate DNA of 3.5–4.0 kilobases (kb),18 sufficient to accommodate the DNA (o3 kb) encoding cytokines and neurotrophins.37 AAV vectors are less toxic than Ad Combination of cochlear implant and gene therapy vectors,39 although damage to cochlear architecture has In addition to treating chemical and noise-induced been observed in a 24-day study.31 Like Ad vectors, there hearing loss, neurotrophin gene therapy can be used is variation in expression profiles among experiments. to improve cochlear implant function. As previously AAVs can infect nearly all cochlear tissue types,38,40 or described, neurotrophins promote the survival of and just the SGCs and stria vascularis.37,39 The reduced delay the degeneration of SGCs. Neurotrophin gene immune response, in particular, makes AAVs attractive therapy performed in conjunction with cochlear implant for further exploration. surgery would likely enhance neurite growth to the cochlear implant. The performance of cochlear implants Herpes simplex viral is contingent on the presence and function of these The herpes simplex virus (HSV) is neurotrophic. In the neurons. The development of higher performing co- cochlea, HSV vectors transfect primarily SGCs.22,41 NT-3 chlear implants would improve the quality of life for stimulates the survival of auditory neurons. One group many deaf children (Figure 3). showed that an HSV/NT-3 vector injected into the scala As previously established, neurotrophins are impor- vestibuli could enhance SGC survival 2 days after low- tant in the development of the inner ear as well as the concentration cisplatin administrations.41 HSV has also survival of adult inner ear sensory cells against exogen- been shown to enter a latent phase in certain neuronal ous damage. Thus, applying stem cells with genes cell types, offering the possibility of stable transfec- knocked-in may prevent or treat inner ear disorders. tion.40,41 A drawback is the pathogenic nature of the Specifically, stem cells with genes for neurotrophins NT- virus and its difficulty of production. Moreover, many 3, BDNF, GDNF, VEGF should be infused into the people have been infected by HSV. HSV research is in a cochlea of deaf animals, to promote in vivo differentiation developmental stage for inner ear applications, but holds of the stem cells into sensory, supporting and neural cells potential in promoting neuronal cell survival. in the cochlea. This is extremely interesting as a potential

Lentiviral Unlike other retroviral vectors, lentiviral vectors (LVs) can infect nondividing cells. LVs transfect a broad range of cells, including neural cells. Like AAVs, LVs have a relatively low inﬂammatory potential. LV-mediated protein expression has been shown in rodent brain for up to 6 months.29 In the inner ear, expression has been detected up to 14 days postinjection, without tissue damage.42 After the injection of LV/GFP into the perilymph, the protein was detected in cells directly Figure 3 Vision of gene therapy and cochlear implant combined therapy.

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