Treatment of peripheral sensorineural hearing loss: gene ... - Nature

Gene Therapy (2004) 11, S51–S56 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt

REVIEW

Treatment of peripheral sensorineural hearing loss: gene therapy M Duan1,2,3,4, F Venail5, N Spencer1 and M Mezzina6 1

Department 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 of irreversible hearing loss, which at present have no cure. Gene therapy may soon be utilized in both the protection and the treatment of these exogenous and endogenous sources of hearing loss. Gene therapy technology is rapidly developing and the inner ear is a particularly feasible model for gene therapy. This review outlines our current understanding of the mechanisms behind deafness and prospects for treat-

ment, discusses the inner ear model in detail and reviews the efforts that have been made in inner ear gene therapy. Finally, the proposed next steps will be discussed. The viral mediated delivery of neurotrophins and antoxidants offers imminent promise in preventing and treating exogenous hearing loss and improving cochlear implant therapy. Gene Therapy (2004) 11, S51–S56. doi:10.1038/sj.gt.3302369

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

Introduction The cochlea contains two types of sensory cells, inner hair cells (IHC) and outer hair cells (OHC), which carry out the transduction of sounds to electrical signals. These signals are transmitted to the auditory pathway of the brain via spiral ganglion neurons (SGCs). Unfortunately, neither of these sensory or primary neural cells have the capacity to regenerate. Thus, any injury to the cochlea can result in an irreversible hearing loss. Despite recent developments in medicine, there are still no clinically useful means for curing hearing disorders and protecting auditory function. It is of paramount importance to develop novel and effective methods of treating both inherited and acquired hearing loss. Gene therapy technology has improved in recent years, making it a promising technique for treating such inner ear disorders. The inner ear holds several unique advantages as a model for gene therapy. Firstly, the cochlea is anatomically well suited for in vivo gene therapy. The relative isolation of the cochlear compartments minimizes unwanted effects of the introduced gene into other tissues. The inner ear is fluidfilled, allowing all functionally important cells to be accessed by a transfection reagent. The concentration and dosage of complexes introduced to the cochlea can easily be modulated with a single injection or longer infusion by an osmotic pump. Cochlear endolymph and perilymph volumes have been characterized in the guinea pig, rat and mouse, so adverse effects of high volume and pressure can be avoided. Correspondence: Dr M Duan, Center for Hearing and Communication Research, Building MI-ENT, Karolinska Hospital, SE-171 76 Stockholm, Sweden

Secondly, various physiological measurement tools have been developed to monitor the function of specific cells (Table 1), and thus assess the efficacy and safety of gene therapy. Cochlear Microphonics (CM) and Otoacoustic emission (OAE) measurements are useful in assessing the state of damage of OHCs. Compound Action Potentials (CAPs) are recorded to check IHC function. Similarly, single unit recordings are used in SGC assessment. More general methods for monitoring cochlear physiology exist as well. Detection of Endocochlear Potentials (EPs) helps to determine the presence of endolymphatic ion balances. A general diagnostics tool is Auditory Brainstem Response (ABR) measurements. Thirdly, the cochlea is unique among sensorineural systems in that so many genes have been recently cloned in the mouse and human. Over 90 different genes have now been identified that affect inner ear development or function, as well as many loci known to be involved in deafness. Between 1996 and 2000, 19 genes involved in nonsyndromic deafness were identified in a larger number of genes implicated in syndromic deafness.1 In addition, a transgenic technique has been demonstrated in shaker-2 mice to correct deafness.2 Finally, there are many possibilities for using gene therapy in the cochlea to treat deafness. Neurotrophin gene therapy is an important example. Neurotrophic factors are essential in the development of the inner ear and in the protection of the adult inner ear sensory cells against ototoxic chemical and noise induced damage.3 Neurotrophin gene therapy is promising both in the protection against exogenous damage and the regeneration after endgenous and exogenous damage. Neurotrophins can be applied through in vivo inoculation of vectors carrying their genes or more indirectly through gene therapy-modified stem cells.

Treatment of peripheral sensorineural hearing loss M Duan et al

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Table 1 Methods for observing various cochlear characteristics post gene therapy inoculation Function

Outer hair cell – cochlear otoacoustic emission Outer hair cell – cochlear microphonics Inner hair cell – compound action potential Spiral ganglion cell – Single unit recording Cochlear fluid – endocochlear potential Auditory nerve – auditory brainstem response (ABR)

Structure

Light microscopy Immunocytochemistry Confocal microscopy

UltrastructureTransmission electron microscopy Scanning electron microscopy

Exogenous and endogenous causes of deafness Many antibiotic and anticancer drugs are ototoxic. Aminoglycoside antibiotics have been successfully used in the treatment of various infectious diseases for more than 40 years. These drugs are in use today, despite of their side effects. Clinical aminoglycoside use can cause severe degeneration of SGCs and hair cells.4 The anticancer drug cisplatin was found to have ototoxic side effects early on in its clinical trials.5 Hearing loss induced by cisplatin is typically strongest at high frequencies (3–8 kHz) in humans, and repeated administrations affect successively lower frequencies accompanied by progressive hearing loss.6 Cisplatin primarily damages OHCs and auditory nerve fibres.7 Noise-induced hearing loss (NIHL) is responsible for one-third of acquired sensorineural hearing loss cases. NIHL can be temporary or permanent. Those who suffer from NIHL also tend to have increased vulnerability to chemical ototoxic insults.8 Approximately one in 800 children are born with a severe to profound genetic-related hearing loss (GRHL). Single-gene defects likely account for the majority of childhood deafness cases.1 The most common cause of GRHL is the GJB2 (Cx26) mutation. Owing to the lack of proliferation in postnatal inner ear sensory epithelia, these problems do not have a cure. Gene therapy is the most promising solution for curing them.

Mechanisms of peripheral hearing loss and prospects for treatment The underlying mechanisms of sensorineural hearing loss are not completely understood. We do, however, have working hypothesies for surmounting these challenges through gene therapy. The following is an outline of likely mechanisms and how they can be prevented and treated.

Reactive oxygen species (ROS) and antioxidants Significant ROS generated in the reduction of oxygen to water include the superoxide anion, hydrogen peroxide and the hydroxyl radical. These ROS are involved in the mechanisms of hearing loss caused by noise trauma,9 Gene Therapy

cisplatin10 and aminoglycoside otoxicity.11 Antioxidants have been shown to protect the cochlea from the damage caused by these factors.9–11 Further evidence exists in particular for superoxide’s role in hearing loss. Superoxide dismutase (Sod) knockout mice have an enhanced susceptibility to noise trauma, resulting in severe NIHL.12 Also, the overexpression of this enzyme protects mice from aminoglycoside ototoxicity in the transgenic model.13

Role of neurotrophic factors in protecting the inner ear Neurotrophic factors are important in inner ear development and adult inner ear protection. Neurotrophin 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 required for the survival of type 2 SGCs innervating OHCs and vestibular hair cells.14 Aminoglycosideinduced degeneration was prevented by the infusion of NT-3 in 90% of adult SGCs.15,16 Neurotrophins NT-3, glial-derived neurotrophic factor (GDNF) and BDNF are important in protection of the cochlea against NIHL.4 Finally, vascular endothelial growth factor (VEGF) is also important in axonal outgrowth and cochlear sensory and supporting cell survival.17 Neurotrophin therapy is promising in the prevention of exogenous hearing loss. The treatment of peripheral hearing disorders with neurotrophic factors had not been widely investigated until very recently. Miller et al18 demonstrated the enhancement of SGC survival after the kanamycin and ethacrynic acid-induced loss of IHCs, through the chronic infusion of BDNF into the cochlea. Recent results showed that the infusion of NT-3 not only protects SGCs from aminoglycoside ototoxicity but also facilitates neurite regrowth.15,16 Moreover, combined BDNF and ciliary neurotrophic factor (CNTF) therapy increases the survival of SGCs when applied as late as 2 weeks following kanamycin treatment.19 This study also used electrical auditory brainstem response measurements to show an enhancement of auditory performance due to neurotrophin treatment of the kanamyicn insult. This is important because the success of cochlear implant treatment depends on the presence of SGCs at the site of the implant. Cochlear implant therapy could be improved by applying neurotrophins to the human cochlea.

Monitoring auditory function after gene therapy Gene delivery systems must not be cytotoxic, and cells must be able to return to a normal physiological state after treatment. Many techniques exist to assess cochlear physiology. First, ABR responses to sound input are used to noninvasively record activity in all the parts of the auditory pathway, allowing for a long-term survey. Using an electrode placed on the round window, the CAP method yields more sensitive results than the ABR. Various OAE measurements noninvasively assess the properties of cochlear sound amplification related to OHC function. All of these techniques can be used to evaluate the recovery of auditory function after an invasive surgery or after gene delivery. Additionally,


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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, lowpotassium 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. 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 ear (Figure 2). A cochleostomy, a small hole in the cochlear bone, can be made for access to the scala tympani. Both of these methods have been shown to deliver drugs effectively without hindering rodent cochlear function.15,16,20 The cochleostomy is advantageous in gene transfer efficacy, while the RW method is less invasive. Low perfusion volumes should be used in the cochleostomy. Recently, the placement of gelfoamsoaked therapeutics on the RW was shown useful in the delivery of certain vectors.21 The vestibular superior semicircular canal can also be accessed via a canalostomy. A cochleostomy can also be made for access to the scala media.

Gene therapy in the inner ear Nonviral Liposomes are the traditional nonviral vector used in inner ear research. They are easy to prepare, can be complexed with DNA of any size, and have a very low risk of insertional mutagenesis. Liposome complexed with LacZ and GFP reporter genes have successfully

Figure 1 Cochlear architecture, highlighting the sensory hair cells of the organ of Corti and the afferent spiral ganglion neurons.

Figure 2 Locating and perfusing the guinea pig round window.

transfected nearly all tissue types of mice and guinea pig cochleae in vivo.22–24 Evidence has not shown clinically useful transfection efficiencies. A broader investigation of vectors and promoters, and the incorporation of nuclear localization sequences and targeting ligands could result in an efficient vector. Better insight into cell membrane composition and nuclear transport may also result in improvements. Other methods such as electroporation25 and the gene gun26 have yielded significant in vitro results but have not been developed for effective use in vivo. Another concept is the nonviral vector with virally derived fusion activity. The hemaglutinating virus of Japan envelope vector recently transfected more than 70 percent of SGCs without affecting ABR performances, after injection to the cisterna magna.27 The injection method raises issues such as dissemination beyond the cochlea,28 but this vector may prove more efficient than other nonviral vectors and safer than viral vectors. This study addressed not only the prevention but also the treatment of kanamycin ototoxicity with the incorporation of the gene for hepatocyte growth factor.

Adenoviral The majority of inner ear gene therapy research has been carried out with replication-deficient (E1 , E3 ) Adenoviral (Ad) vectors, which can be generated at high concentrations29and can accommodate large (8 kb) fragments of DNA. They are efficient in infecting many inner ear cell types in vivo, including SGCs.4,17 The replication defective (E1 E3 , pol ) 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 from gentamicin ototoxicity. It has also been shown to protect OHCs from gentamicin ototoxicity,30 in a study Gene Therapy


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where Ad infection of hair cells was not shown. The reason for the protective effect is unknown, but may be attributed to the secretion of proteins. Combined cytokine transforming growth factor (TGF)-b1/GDNF Ad gene therapy improves this protection, but with accompanying fibrosis.32 Ad-GDNF delivery also protects IHCs after an acute cessation of blood supply.33 Antioxidant gene therapy has also proven useful in mouse models.34 Neurotrophin and antioxidant gene therapy hold promise in treating deafness.4 The major drawback is the capsid-induced immune response,35 typically causing the clearance of infected 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 immune responses have been carried out with two types of immunosuppressants: T-lymphocyte costimulator inhibitors and the anti-inflammatory steroids. Both types of suppressants can inhibit the Ad-immune response and allow the production of the second Ad-delivered transgene.36 Care must be taken, however, to avoid infection in human patients during immunosuppressant therapy.

Adeno-associated viral Adeno-associated viral (AAV) vectors37 elicit a much less potent immune response than Ad vectors. In fact, AAVs have been found to mediate gene expression for up to 6 months, albeit at decreased levels.38 AAV vectors can 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 vectors,39 although damage to cochlear architecture has been observed in a 24-day study.31 Like Ad vectors, there is variation in expression profiles among experiments. AAVs can infect nearly all cochlear tissue types,38,40 or just the SGCs and stria vascularis.37,39 The reduced immune response, in particular, makes AAVs attractive for further exploration.

accessible by the fluid. Less accessible cells such as the spiral ganglia and glial cells have been transfected in vitro.42 Based on these early studies, the potential uses for this vector involve the secretion of growth factors and antibiotics into the perilymph. The possibility of insertional mutagenesis must be carefully studied in the inner ear before seriously considering this vector for clinical use. The insertion of genes into the chromosome is also advantageous, however, in the potential treatment of genetic hearing loss. For this reason, LV gene therapy holds promise in treating both endogenous and exogenous causes of deafness.

The Future Treating exogenous deafness through viral-mediated expression of secreted products Ad, AAV and LV vectors effectively transfer genes into the inner ear in vivo. AAV and LV vectors have shown lengthy transgene expression times and low toxicity. Methods have been developed for the inoculation of vectors into the cochlea as well as the detection of gene products and the assessment of cochlear function. It is particularly promising to influse AAV and LV vectors carrying the neurotrophin and antioxidant enzyme genes NT-3, BDNF, GDNF, VEGF and Sod to the guinea pig and mouse cochleae in treating chemical and noise-induced inner ear disorders.

Herpes simplex viral The herpes simplex virus (HSV) is neurotrophic. In the cochlea, HSV vectors transfect primarily SGCs.22,41 NT-3 stimulates the survival of auditory neurons. One group showed that an HSV/NT-3 vector injected into the scala vestibuli could enhance SGC survival 2 days after lowconcentration cisplatin administrations.41 HSV has also been shown to enter a latent phase in certain neuronal cell types, offering the possibility of stable transfection.40,41 A drawback is the pathogenic nature of the virus and its difficulty of production. Moreover, many people have been infected by HSV. HSV research is in a developmental stage for inner ear applications, but holds potential in promoting neuronal cell survival.

Combination of cochlear implant and gene therapy In addition to treating chemical and noise-induced hearing loss, neurotrophin gene therapy can be used to improve cochlear implant function. As previously described, neurotrophins promote the survival of and delay the degeneration of SGCs. Neurotrophin gene therapy performed in conjunction with cochlear implant surgery would likely enhance neurite growth to the cochlear implant. The performance of cochlear implants is contingent on the presence and function of these neurons. The development of higher performing cochlear implants would improve the quality of life for many deaf children (Figure 3). As previously established, neurotrophins are important in the development of the inner ear as well as the survival of adult inner ear sensory cells against exogenous damage. Thus, applying stem cells with genes knocked-in may prevent or treat inner ear disorders. Specifically, stem cells with genes for neurotrophins NT3, BDNF, GDNF, VEGF should be infused into the cochlea of deaf animals, to promote in vivo differentiation of the stem cells into sensory, supporting and neural cells 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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Figure 4 Schematic overview of the application of stem cell gene therapy to the improvement of hearing.

method for overcoming the lack of proliferation of these cells in the inner ear (Figure 4) and treating deafness by many causes.

Acknowledgements This work was supported by the Swedish Research Council, the Foundation Tysta Skolan, Stiftelsen Clas Groschinskys Minnesfond, Educational Department of Anhui Province, Department of Science and technology, Anhui Province, China.

References 1 Steel K, Kros C. A genetic approach to understanding auditory function. Nat Genet 2001; 27: 143–149. 2 Probst FJ et al. Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 1998; 280: 1444–1447. 3 Duan ML et al. Framtida bot fo¨r ho¨rselskador? Geneterapi och implamtation av stamceller mo¨jliga nya behandlingsva¨gar. La¨kartidningen 2000; 97: 1106–1112. 4 Duan ML et al. Protection and treatment of sensorineural hearing disorders caused by exogenous factors: experimental findings and potential clinical application. Hear Res 2002; 169: 169–178. 5 Schweitzer BD. Ototoxicity of chemotherapeutic agents. Otolaryngol Clin North Am 1993; 26: 759–789. 6 Laurell G. Ototoxicity of the anticancer drug cisplatin-clinical and experimental aspects. Thesis 1991, Karolinska Institutet. 7 Wang J et al. Local application of sodium thiosulfate prevents cisplatin-induced hearing loss in the guinea pig. J Neuropharmacol 2003; 45: 380–393. 8 Johnson AC, Nylen P, Borg E, Hoglund G. Sequence of exposure to noise and toluene can determine loss of auditory sensitivity in the rat. Acta Otolaryngol 1991; 109: 34–40. 9 Duan ML et al. Dose and time-dependent protection of the antioxidant N-L-acetylcysteine against impulse noise trauma. Hear Res 2004; 192: 1–9. 10 Rybak LP et al. Effect of protective agents against cisplatin ototoxicity. Am J Otol 2000; 21: 513–520. 11 Sha SH, Schacht J. Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: Dmethionine is a potential protectant. Hear Res 2000; 142: 34–40. 12 Ohlemiller KK et al. Targeted deletion of the cytosolic Cu/ Zn-superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss. Audiol Neurootol 1999; 4: 237–246.

13 Sha S, Zajic G, Epstein C, Schacht J. Overexpression of copper/ zinc superoxide dismutase protects from kanamycin-induced hearing loss. Audiol Neurootol 2001; 6: 117–123. 14 Ernfors P, Van De Water TR, Loring J, Jaenisch R. Complementary roles of BDNF and NT-3 in auditory and vestibular development. Neuron 1995; 14: 1153–1164. 15 Ernfors P, Duan ML, Elshamy WM, Canlon B. Protection of auditory neurons from aminoglycoside toxicity by neurotrophin-3. Nat Med 1996; 2: 463–467. 16 Duan ML, Agerman K, Ernfors P, Canlon B. Complementary roles of neurotrophin 3 and a N-methyl-D-aspartate antagonist in the protection of noise and aminoglycoside-induced ototoxicity. Proc Natl Acad Sci USA 2000; 97: 7597–7602. 17 Hess A et al. In vitro activation of extracellular signal-regulated kinase 1/2 in the inner ear of guinea pigs. Brain Res 2002; 956: 236–245. 18 Miller JM et al. Neurotrophins can enhance spiral ganglion cell survival after inner hair cell loss. Int J Dev Neurosci 1997; 15: 631–643. 19 Shinohara T et al. Neurotrophic factor intervention restores auditory function in deafened animals. Proc Natl Acad Sci USA 2002; 99: 1657–1660. 20 Chen Z et al. Acute treatment of noise trauma with local caroverine application in the guinea pig. Acta Otolaryngol 2003; 123: 905–909. 21 Jero J et al. Cochlear gene delivery through an intact round window membrane in mouse. Hum Gene Ther 2001; 12: 539–548. 22 Staecker H, Li D, O’Malley B, Van De Water T. Gene expression in the mammalian cochlea: a study of multiple vector systems. Acta Otolaryngol 2001; 121: 157–163. 23 Wareing M et al. Cationic liposome mediated transgene expression in the cochlea. Hear Res 1999; 128: 61–69. 24 Jero J, Tseng CJ, Mhatre AN, Lalwani AK. A surgical approach appropriate for targeted cochlear gene therapy in the mouse. Hear Res 2001; 151: 106–114. 25 Zheng JL, Gao WQ. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 2000; 3: 580–586. 26 Schneider ME, Belyantseva IA, Azevedo RB, Kachar B. Rapid renewal of auditory hair bundles. Nature 2002; 418: 837–838. 27 Oshima K et al. Intrathecal injection of HJV-E containing HGF gene to cerebrospinal fluid can prevent and ameliorate hearing impairment in rats. FASEB J 2004; 18: 212–214. 28 Stover T, Yagi M, Raphael Y. Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer. Gene Therapy 2000; 7: 377–383. 29 Verma IM, Somia N. Gene therapy – promises, problems and prospects. Nature 1997; 389: 239–242. 30 Luebke AE, Steiger JD, Hodges BL, Amalfitano A. A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function. Gene Therapy 2001; 8: 789–794. 31 Yagi M et al. Spiral ganglion neurons are protected from degeneration by GDNF therapy. J Assoc Res Otolaryngol 2000; 1: 315–325. 32 Kawamoto K et al. Hearing and hair cells are protected by adenoviral gene therapy with TGF-B1 and GDNF. Mol Ther 2003; 7: 484–492. 33 Hakuba N et al. Adenovirus-mediated overexpression of a gene prevents hearing loss and progressive inner hair cell loss after transient cochlear ischemia in gerbils. Gene Therapy 2003; 10: 426–433. 34 Agrawal RS et al. Pre-emptive gene therapy using recombinant adeno-associated virus delivery of extracellular superoxide dismutase protects heart against ischemic reperfusion injury, improves ventricular function and prolongs survival. Gene Therapy 2004; 11: 962–969. 35 Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Genet Rev 2003; 4: 346–358. Gene Therapy


S56 36 Blair E. Adenoviral vectors, breaking a barrier to gene therapy? Gene therapy 2004; 11: 229–230. 37 Duan ML et al. Adenoviral and adeno-associated viral vector mediated gene transfer in the guinea pig cochlea. NeuroReport 2002; 13: 1295–1299. 38 Lalwani AK et al. Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus. Gene Therapy 1998; 5: 277–281. 39 Luebke AE, Foster PK, Muller CD, Peel AL. Cochlear function and transgene expressio in the guinea pig cochlea, using

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adenovirus- and adeno-associated virus-directed gene transfer. Hum Gene Ther 2001; 12: 773–781. 40 Lalwani AK, Mhatre AN. Cochlear gene therapy. Ear Hearing 2004; 24: 342–348. 41 Bowers WJ et al. Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea. Mol Ther 2002; 6: 12–18. 42 Han JJ et al. Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector. Hum Gene Ther 1999; 10: 1867–1873.