mediated cochlear gene transfer - Nature

Gene Therapy (2000) 7, 377–383  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

VIRAL TRANSFER TECHNOLOGY

RESEARCH ARTICLE

Transduction of the contralateral ear after adenovirusmediated cochlear gene transfer T Sto¨ver1,2, M Yagi1,3 and Y Raphael1 1

Kresge Hearing Research Institute, Department of Otolaryngology, The University of Michigan Medical School, Ann Arbor, MI, USA; 2Department of Otolaryngology, Medizinische Hochschule Hannover, Hannover, Germany; and 3Department of Otolaryngology, Kansai Medical University, Osaka, Japan

Cochlear gene transfer is a promising new approach for inner ear therapy. Previous studies have demonstrated hair cell protection with cochlear gene transfer not only in the inoculated, but also in the uninoculated ear. To characterize the kinetics of viral spread, we investigated the extent of transgene expression in the contralateral (uninoculated) cochlea after unilateral adenoviral cochlear gene transfer. We used a lacZ reporter gene vector, and demonstrated spread of the adenovirus into the cerebrospinal fluid (CSF) after cochlear inoculation of 25 ␮l viral vector. Direct virus application into the CSF resulted in transduction of both

cochleae, whereas virus inoculation into the bloodstream did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virus-mediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Our results showed a functional communication between the CSF and the perilymphatic space of the inner ear, that is not only of importance for otological gene transfer, but also for CNS gene transfer. Gene Therapy (2000) 7, 377–383.

Keywords: adenovirus; gene transfer; cochlea; guinea pig

Introduction A variety of pathological processes may lead to cochlear and vestibular impairments. Prevention or correction of such pathological processes is, thus far, clinically restricted to the systemic application of substances. Cochlear gene transfer provides both an opportunity for localized administration and the potential for molecular-based interventions. Such intervention may facilitate overexpression of certain gene products that may influence repair and regeneration in the inner ear epithelia. For instance, manipulating levels of retinoic acid in cultured developing cochleae and knocking out the p27Kip1 gene have been shown to lead to overproduction of hair cells.1–3 The former example deals with a secreted (diffusible) gene product and can be done in the mature cochlea. The latter example, p27Kip1 knock out, can presently be accomplished only at the germline intervention level. However, once gene therapy in somatic cells shuts down the expression of specific genes, similar results can be accomplished in a tissue-specific manner. Such interventions are particularly exciting for clinical applications as they would provide a treatment of inner ear pathologies that currently have no cure. There are several possible ways to deliver transgenes into tissues. Nonviral vectors pose little or no risk of immune response, but their transduction efficiency is

Correspondence: Y Raphael, MSRB 3, Room 9303, 1150 W. Medical Center Drive, Ann Arbor, MI 48109–0648, USA Received 29 July 1999; accepted 28 October 1999

low. In contrast, viral vectors have been shown to be effective vehicles for gene transfer. Several viral vectors have been used for experimental inner ear gene transfer.4 Among the most promising are the adenovirus,5,6 the adeno-associated virus,7 the herpes simplex virus,8 and the retrovirus.9 Cochlear gene transfer for overexpression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) showed protective effects on hair cells and spiral ganglion cells.10,11 These studies also demonstrated the potential for cochlear gene therapy for therapeutic applications. Following unilateral adeno-associated virus gene transfer into guinea pig cochlea, Lalwani et al12,13 observed transduction of vestibular cells of the uninoculated (contralateral) cochlea. They speculated that the viral vector was transferred to the uninoculated ear via the bloodstream, the bone marrow, or the CSF. Contralateral transgene expression following virus-mediated gene transfer has not been reported elsewhere. However, a trend for the contralateral protective effects of GDNF has been observed.10 If the contralateral effect is true, protection experiments based on a comparison between the inoculated and contralateral sides could underestimate the degree of protection, since the contralateral ear might also have been treated and protected. The mechanism for the contralateral protection is unclear. Therefore, the first goal of this study was to characterize the contralateral effects after unilateral cochlear gene transfer. Specifically, we wished to determine if contralateral transgene expression can occur after adenovirus inoculation of the guinea pig ear and, if so, to elucidate the route of migration of the viral particles to the contralateral ear.

Transduction of the contralateral cochlea T Sto¨ver et al

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We chose an adenoviral vector system, encoding the reporter gene lacZ, to address these questions. Inner ear inoculation with the adenoviral vector is well established5,14 and it has been demonstrated to be therapeutically effective on hair cells.10 As third-generation adenovirus vectors are being developed with reduced immunogenicity and increased duration of gene expression, these vectors are potential candidates for cochlear gene transfer in clinical trials. The second goal of this study was to determine other important features of virus-mediated cochlear gene transfer in the cochlea. Without doubt, clinical application will require detailed information on the possible side-effects of viral vehicles used for cochlear gene transfer. Determination of the transgene distribution after inoculation with increased amounts of viral vector is of great interest as no information is currently available on the maximal volumes of virus solution applicable to the inner ear as a bolus injection. Evaluation of the resulting transgene distribution and identification of the cell types susceptible to the viral vector after inoculation with increased volumes of vector have to occur before gene transfer can be practically used in clinical trials. Other issues to be addressed include unintended transfection of nontargeted tissues with increased virus volume and possible changes in vector cell specificity. We demonstrate that cochlear expression of lacZ was detectable as soon as 6 h after virus inoculation. Contralateral transgene expression was volume dependent. It was observed after an inoculation with 25 ␮l, but not after a 5 ␮l inoculation into the cochlea. No systemic transgene expression was found after cochlear inoculation even with the increased volume of the viral vector, whereas expression in liver and spleen was observed after inoculation into the bloodstream and the CSF. Inoculation into the cranial CSF resulted in detection of lacZ-positive cells in the cochlear opening of the cochlear aqueduct bilaterally, implicating the CSF aqueduct as the route for ear to ear vector transfer. Finally, application of the viral vector into the lumbar region of the CSF space was also identified as a potential route for delivering vectors into the cochlea.

Results Cochlear inoculation of increased vector volume results in transduction of the contralateral ear Cochlear gene transfer via the cochleostomy with 5 ␮l of the adenoviral vector, carrying the reporter gene lacZ, resulted in the transduction of cochlear cells only in the inoculated left ear (n = 6). Transfected cells were localized in the scala tympani of the basal and second turn. After cochlear inoculation with 25 ␮l (n = 6) of adenoviral solution, lacZ-positive cells were observed in both the inoculated (Figure 1A) and the uninoculated (contralateral) cochleae (Figure 1B and C). Transduced cells in the uninoculated contralateral (right) ear were localized predominantly in the scala tympani, at the opening of the perilymphatic aqueduct. LacZ-positive cells were also found on the basal surface of the brain following a 25 ␮l cochlear inoculation (Figure 2A). No transduction of the brain surface or the contralateral cochlea was detected following a 5 ␮l viral inoculation (n = 6) (Table 1). Gene Therapy

Transduced cochlear cells are detectable 6 h after gene transfer Time-course experiments were performed with cochlear inoculations of 25 ␮l adenoviral vector into the left cochlea. Transfected cochlear cells could be detected 6 h after inoculation. None of the animals killed from 30 min to 5.5 h after cochlear gene transfer showed lacZ-positive cells. In animals killed 6 h and 10 h after inoculation, lacZ-positive cells were identified in the scala tympani of both ears. Histological sections obtained from these specimens identified lacZ-positive cells predominantly in connective tissue cells within the spiral ligament, mesothelial cells beneath the organ of Corti, and fibrocyctes lining the perilymphatic fluid spaces (Figure 1D). Furthermore, in some specimens, we found lacZ-staining in Hensen cells and pillar cells (Figure 1E) and also in the region of Rosenthal’s canal. The transfected cells in Rosenthal’s canal were predominantly Schwann cells, lying between spiral ganglion cells (Figure 1F). Six (and 10) h after virus inoculation into the scala tympani, we also observed staining of the middle ear mucosa in the inoculated side (data not shown). The transduction of the contralateral cochlea is mediated by the cochlear aqueduct To determine if the route of transduction for the contralateral cochlear cells was via the blood or CSF mediation, 25 ␮l of adenoviral solution was injected directly into the jugular vein or into the CSF space of the skull. No lacZpositive cells were found on the brain surface or in cochlear tissue of any systemically inoculated animal (n = 4). In contrast, lacZ-positive cells were identified in both cochleae, mostly in the basal and the second cochlear turns, and on the brain surface in all animals inoculated via the CSF (n = 6). Careful inspection of the cochlear tissue revealed a predominant cluster region of lacZ-positive cells close to the ‘hook’ region of the cochlea (Figure 3A and B). This region represents anatomically the opening of the cochlear aqueduct that connects the CSF space with the perilymphatic space of the scala tympani. While 5 ␮l of the viral vector applied to the cochlea were not sufficient to induce a contralateral gene transfer effect, 25 ␮l of the virus solution applied to the cochlea or the CSF space induced a response. To investigate further viral passage through the cochlear aqueduct in guinea pigs, we injected 200 ␮l of the adenoviral vector into the CSF space of the lumbar region via a lumbar puncture (n = 2). We found that one of the animals showed lacZ-positive cells in both cochleae. In this animal, the brain surface was also positively stained for lacZ. The spinal cord was lacZ-positive in both animals. Systemic expression of lacZ-positive cells after cochlear, blood, or CSF inoculation Following cochlear inoculation with 5 ␮l of vector solution (n = 6), we did not detect any systemic expression of lacZ in any organ (liver, spleen, lung, kidney or brain) other than the inoculated ear. After injection of 25 ␮l of vector solution into the cochlea (n = 6), we did not find expression of lacZ outside the above described basal brain region and the contralateral ear. Middle ear staining was observed with 5 ␮l and 25 ␮l inoculation of the cochlea only at the site of inoculation. Virus application directly to the CSF (25 ␮l), however, led to the systemic


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Figure 1 (A) A stereo-micrograph of the left cochlea after cochlear inoculation of the left ear with 25 ␮L adenoviral vector. After X-gal staining, transduced (blue) cells are located in first and second turns of the scala tympani (bar: 1 mm). (B) A stereo-micrograph of the right cochlea after cochlear inoculation of the contralateral (left) ear with 25 ␮l adenoviral vector. After X-gal staining, transduced cells are located predominately in the scala tympani around the ‘hook’ region of the basal turn of the cochlea. The opening of the cochlear aqueduct into the scala tympani is indicated with a hair (arrow) that has been inserted into the duct lumen (bar: 1 mm). C: A stereo-micrograph of the same specimen as (B) (higher magnification) showing the area with the highest density of lacZ-positive cells at the opening of the cochlear aqueduct into the scala tympani. A hair (arrow) has been placed inside the cochlear aqueduct to indicate the location of its opening into the scala tympani (bar: 1.5 mm). D: A lightmicroscopic (LM) section of the organ of Corti after 25 ␮l adenovirus inoculation into the cochlea. Predominantly mesothelial cells (m) beneath the organ of Corti and the fibrocyctes (f) lining the perilymphatic fluid spaces are transduced by the adenoviral vector. The framed box indicates the area of (E) (bar: 100 ␮m). (E) A LM section from the same region as shown in (D) at higher magnification. ␤-galactosidase could be detected in Hensen cells (h) and pillar cells (p) of the organ of Corti (bar: 20 ␮m). (F) A LM section of a left cochlea (Rosenthal’s canal) after cochlear inoculation with 25 ␮l adenoviral vector. LacZ-positive cells (stained blue) are located between spiral ganglion cells and identified as Schwann cells (bar: 20 ␮m).

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Table 1 Localization of lacZ-positive cells according to inoculation side and volume of adenoviral vector used Localization of lacZpositive cells

Inoculated cochlea Non-inoculated cochlea Middle ear Brain surface Liver Spleen Lung Kidney

Adenovirus inoculation volume and site 5 ␮l cochlea (n = 6)

25 ␮l cochlea (n = 6)

25 ␮l blood (n = 4)

25 ␮l CSF (n = 6)

yes no yes no no no no no

yes yes yes yes no no no no

no no no no yes yes no no

yes yes no yes yes yes no no

expression of lacZ in both the liver and spleen (n = 6) but not in the middle ear space or in lung or kidney tissue. Virus application (25 ␮l) directly into the bloodstream via the jugular vein resulted in even stronger expression of lacZ in the liver (Figure 2B and C) and the spleen (Figure 2D and E), whereas no expression was found in the cochlea, middle ear space, cells of the brain surface, the lung, or the kidney (n = 4). These results are summarized in Table 1.

Discussion Following inoculation of the left cochleae with the adenoviral vector, contralateral transfection of cochlear cells was found. The contralateral transfection appeared to be volume dependent, since transfection of the right (contralateral) ear was restricted to experiments in which 25 ␮l of vector solution was inoculated into the left coch-

Figure 2 (A) A stereo-micrograph of the basal brain surface after cochlear inoculation with 25 ␮l adenoviral vector and X-gal staining. LacZ-positive cells (blue stained) are located on the basal brain surface and on blood vessels. Stereo-optical inspection clearly showed blue-colored particles spread along the outside of the blood vessel walls (arrow) (bar: 1.5 mm). (B) A stereo-micrograph of X-gal stained liver of an animal intravenously inoculated with 25 ␮l adenoviral vector. LacZ-positive cells (blue stained) are uniformly distributed throughout the liver (bar: 0.5 mm). (C) A LM section of the liver of an animal intravenously inoculated with 25 ␮l adenoviral vector. LacZ-positive cells were identified as hepatocytes (arrows) (bar: 20 ␮m). (D) A stereo-micrograph of X-gal stained spleen of an animal intravenously inoculated with 25 ␮l adenoviral vector. LacZ-positive cells (blue stained) are distributed throughout the organ (bar: 0.5 mm). (E) A LM section of the spleen of an animal intravenously inoculated with 25 ␮l adenoviral vector. LacZ-positive cells appear like reticular cells in the spleen cords (arrows) (bar: 20 ␮m).

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Figure 3 (A) A stereo-micrograph of a left cochlea after CSF inoculation with 25 ␮l adenoviral vector and X-gal staining. Note that the lacZ-positive cells are located predominantly (dark blue staining) around the ‘hook’ region in the scala tymani of the cochlea. This region represents the opening of the cochlear aqueduct into the scala tympani. Blue cells are also found in the second turn of the cochlea (bar: 1 mm). (B) A stereo-micrograph of the right cochlea of the same animal as shown in (A). After CSF inoculation with 25 ␮l adenoviral vector and X-gal staining, lacZ-positive cells are located predominantly around the ‘hook’ region of the basal turn (bar: 1 mm).


lea. Contralateral transfection was not observed with a small volume (5 ␮l) adenovirus inoculation. A bilateral lack of transfected cells following a systemic intravenous inoculation ruled out the blood-borne transmission of vector for intercochlear spread of virus. Inoculation of the viral vector into the CSF led to a bilateral cochlear transfection, manifested mainly in the mesothelial cells in the scala tympani near the cochlear aqueduct opening. LacZpositive cells were also found in the surface of the brain following inoculation with 25 ␮l of viral vector into the cochlea or the CSF. These results provide strong evidence that the cochlear aqueduct is the mediating route for intercochlear communication leading to contralateral vector spread. Our data conclusively demonstrate contralateral transfection of cochlear cells with adenovirus vectors as early as 6 h after inoculation. Transfection of contralateral cells might have occurred earlier, considering that the expression of ␤-galactosidase may require several hours to manifest. Contralateral transfection has previously been observed with a smaller viral vector, the adenoassociated virus.12,13 These authors speculated that mediation of the vector to the uninoculated ear was via the blood, bone marrow, or CSF. As the perilymphatic fluids are not compressible and perilymph leakage was not observed during our surgical procedures, fluid displacement inside the cochlea had to take place during the application of additional volume into the perilymphatic space. This is even more obvious, considering that the viral volume (25 ␮l) applied to the perilymphatic space exceeded the volume of the perilymphatic space (16 ␮l).15 We speculate that fluid application to the cochlea results in a displacement of perilymphatic fluid via the cochlear aqueduct into the CSF. Our findings support this hypothesis on four different points. First, we demonstrated that viral vector inoculation into one cochlea leads to the detection of cells expressing the transgene in the contralateral cochlea. Second, in these animals we found the cells on the brain surface consistently transduced, showing the presence of vector in the CSF space, which is continuous with the cochlear aqueduct. Third, when vector was applied directly to the CSF, it not only transduced the brain surface, it also spread into the scala tympani of both cochleae. Fourth, after CSF inoculation, transduced cells were located predominantly around the opening of the cochlear aqueduct near the ‘hook’ region of the scala tympani of both cochleae. This distribution pattern was almost symmetrical and identical to the distribution found in the uninoculated (right) ear after cochlear inoculation with 25 ␮l of the vector. Finally, we ruled out blood-borne transduction of cochlear cells as the cause of contralateral gene transfer as no cochlear gene transfer was observed after direct inoculation of the vector into the bloodstream. These data strongly suggest that the cochlear aqueduct and the CSF facilitate inter-aural vector spread (see Figure 4 for a schematic). These findings are of practical importance for the clinical application of gene therapy in the inner ear. The connection between both cochleae may account for several unexplained contralateral gene transfer effects after unilateral inoculation. An adenovirus vector encoding the human GDNF gene was shown to protect the inoculated (left) ears from ototoxic drugs and, in addition, to afford some protection to the contralateral (uninoculated) ear.10 Due to the contralateral protection, it is likely that

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Figure 4 (A) A schematic of the ear anatomy, emphasizing the connection between the perilymphatic space of the cochlea (scala tympani) and the CSF space. (B) The same schematic as (A), with red arrows indicating the route of vector spread after inoculation of 25 ␮l viral vector into the scala tympani of the cochlea. Virus spread is from the perilymph of the scala tympani via the cochlear aqueduct to the CSF, and further via the contralateral cochlear aqueduct to the opposite cochlea. (C) The red arrows indicate the route of vector spread after direct inoculation of 25 ␮L viral solution into the CSF. The viral vector is distributed within the CSF and reaches the perilymphatic space of both cochleae via the cochlear aqueducts.

assessing protective effects (following bilateral insults) by comparing the inoculated with the uninoculated ear would underestimate the degree of protection. The implication of the contralateral transfection for gene therapy, as a consequence of exceeding a certain inoculation volume, is spread of the viral vector or diffusion of secreted proteins beyond the inoculated cochlea. Therefore, localized cochlear gene transfer can be achieved only if inoculation is performed with relatively low volumes of viral vector. On the positive side, our findings imply that the cochlear aqueduct might be used as a route for gene transfer of the cochlea. Our results after inoculation of the vector directly into the CSF demonstrate that, in principle, gene transfer in the cochlea can be accomplished without directly manipulating the cochlea. In the present animal experiments, we have shown that even after application of the vector to the CSF via a lumbar puncture, transfected cells were observed in both cochleae. These results might lead to a new approach for inner ear gene transfer or drug application, both from the CSF to the inner ear and from the inner ear to the CSF. The cochlear aqueduct as an accessible gate to the cochlea via the CSF has implications beyond the area of Gene Therapy


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gene transfer. Bilateral deafness after meningitis, particularly in young children with bacterial meningitis or viral mumps meningitis, might progress via the cochlear aqueduct to the cochlear space.16 Clinically, it has been found that an elevated CSF pressure is a predicting factor for the occurrence of deafness after bacterial meningitis.16 Since we have demonstrated the functional connection between the cochleae and the CSF space via the cochlear aqueduct, pathological processes occurring at the CSF level, eg pneumococcal meningitis,17 might be considered as an explanation for the bilateral effects on the cochlear tissue. Localization of lacZ-positive cells in the cochlear tissue after inoculation of 25 ␮l of the adenoviral vector showed transduction predominantly of mesothelial cells beneath the organ of Corti and fibrocyctes lining the perilymphatic fluid spaces (Figure 1D). X-gal staining was also found in Hensen cells, pillar cells (Figure 1E), and Schwann cells in Rosenthal’s canal (Figure 1F). Thus, comparison between the types of cells transduced with a 5 ␮l or 25 ␮l vector revealed a higher quantity and larger spread of the transfection with the higher volume of vector. In addition, the higher volume of vector inoculation resulted in transduction of two types of supporting cells in the organ of Corti, which did not express the reporter transgene following the lower volume of inoculation. The transduction of supporting cells is important and exciting because of their key role for repair and regeneration,18–20 presenting these cells as a main target for gene transfer in the inner ear. Systemic expression of lacZ in the liver and spleen was found after CSF and intravenous inoculation of the adenovirus (25 ␮l), but not after cochlear inoculation with the same increased volume of virus solution (25 ␮l), indicating less vector being distributed into the CSF after cochlear inoculation compared with direct CSF inoculation. No lacZ expression was found in lung or kidney tissue. Cochlear inoculations with lower amounts of the adenoviral vector (5 ␮l), exclusively and consistently showed that transduction was restricted to the inoculated (left) ear. Therefore, the data demonstrate that small volumes (5 ␮l) of vector can be safely applied for cochlear gene therapy (as a bolus injection), without risking vector spread outside the inoculated cochlea. In our experiments, transfected cells were also found in the middle ear space under some experimental approaches. This occurred only in animals after direct cochlear inoculation, but not after blood or CSF inoculation. No apparent difference was noted in the amount of middle ear staining after either 5 ␮l or 25 ␮l vector inoculation. The perilymphatic flow rate has been shown to increase by about two orders of magnitude following an experimentally induced fistula.21 Therefore, an increased perilymph flow as a result of the inoculation may increase the risk of viral spread into the middle ear. Perilymph leakage may be due to an incomplete seal at the injection site. Even though a leak was undetected during the procedure, the presence of transfected middle ear mucosal cells suggests that a leak probably occurred. Unwanted middle ear transfection involves the potential risk of vector spreading via the Eustachian tube to the airways and to the digestive tract. However, transfection of middle ear cells also presents therapeutic opportunities that may be harnessed for interventions in the

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middle ear mucosa, as has been demonstrated by Mondain et al.22 In conclusion, we detected contralateral cochlear transgene expression. The contralateral spread was dependent on the volume of inoculated vector solution. Virus inoculation into the CSF resulted in bilateral cochlear transduction, whereas systemic virus inoculation did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virusmediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Furthermore, our data provide evidence for a close functional communication between the CSF and the perilymphatic space of the inner ear, a finding that is of importance for otological gene transfer as well as CNS gene therapy.

Materials and methods Adenoviral vector To perform cochlear gene transfer experiments, we used a replication-deficient adenoviral vector (E1A/B and part of E3 deleted) based on the human adenoviral backbone (serotype 5). The vector carried the reporter gene lacZ (Ad. CMVlacZ), encoding E. coli ␤-galactosidase, driven by the cytomegalovirus promoter. The viral vector was constructed as has been described previously23 and obtained from the University of Michigan Vector Core. The vector was stored at −80°C until use. It was diluted with Ringers’ solution (145 mm NaCl, 2.7 mm KCl, 2 mm MgSO4, 1.2 mm CaCl2, 5 mm Hepes) to a final concentration of 1010 plaque-forming units (p.f.u.)/ml immediately before inoculation. In experiments comparing virus inoculation with 5 ␮l and 25 ␮l, the same batch of adenoviral vector was used. Animal procedures and gene transfer All animal experiments were approved by the University Committee on the Use and Care of Animals at the University of Michigan and were performed using accepted veterinary standards. Guinea pigs of either sex (Murphy Breeding Laboratory, Plainfield, IN, USA), weighing 237– 387 g, were used. Viral administration into the cochlea was via a cochleostomy, as previously described.24 The animals were inoculated with either 5 ␮l (n = 6) or 25 ␮l (n = 6) adenoviral solution into the scala tympani of the left cochlea. A movement of the round window membrane could be observed during inoculation of 25 ␮l performed over approximately 3 min. This movement was used to monitor the injection velocity. Application of the viral vector into the bloodstream (n = 4) was performed with a 25 ␮l virus solution injected directly into the right jugular vein. Virus application into the CSF was performed with 25 ␮l virus solution injected into the subdural space (n = 6). For adenovirus application to the CSF, animals were anesthetized (xylazine 10 mg/kg i.m. and ketamine 40 mg/kg i.m.), and a 3 × 3 mm piece of skull bone, near to the bregma, was carefully removed with a diamond burr. The dura was incised and a 5 cm piece of vinyl tubing V/4 (Scientific Commodities Incorporation, Lake Havasu City, AZ, USA) was inserted about 4 mm into the subdural space. To prevent CSF leakage, the cannula was


secured to the skull with carboxylate cement (Durelon, ESPE, Germany). After the cement dried (approximately 10 min), the viral solution was injected into the CSF. The cannula was left in place for 10 min and then withdrawn. The skull opening was sealed with bone wax (Ethicon, Summerville, NJ, USA) and the skin sutured in two layers. Alternatively, a lumbar puncture using a 25-gauge needle (n = 2) was performed to inject 200 ␮l of virus solution into the CSF space. Animals were carefully monitored postsurgically for discomfort, head-tilt, feeding, and general activity level. Recovery was uneventful in all animals. The animals were decapitated 5 days after vector inoculation and temporal bones, lung, spleen, liver, kidneys, and brain tissue were removed.

Tissue processing Collected tissue samples (temporal bones, brain, lung, spleen, liver and kidney) were fixed with 4% paraformaldehyde for 2 h at 4°C and then rinsed twice for 10 min with phosphate-buffered saline (PBS). To detect expression of the transgenic ␤-galactosidase, the tissues were incubated overnight at 37°C in X-gal solution (5bromo-4-chloro-3-indolyl-␤-d-galactoside) as described previously.23 Following a PBS rinse, the tissues were photographed under a dissection microscope (Wild MZ12). Tissues were sectioned for histological localization of lacZ-positive cells. The cochleae were decalcified in 3% EDTA with 0.25% glutaraldehyde for about 10 days. Tissues were then dehydrated in an ethanol series and embedded in JB-4 media (Electron Microscopy Sciences, Washington, PA, USA). Sections obtained at the midmodiolar area (4 ␮m thick) were stained with toluidinblue (1%) or eosin-red (0.5%) and cover-slipped with Permount (Fisher Scientific, Springfield, NJ, USA). Gene transfer time-course For the time-course experiment, cochlear gene transfer via a cochleostomy24 was performed with 25 ␮l of the adenoviral solution in the 17 guinea pigs, as described above. After vector inoculation, the animals were killed after 30 min, 1 h, 2 h, 3 h and 3.5 h (one animal each), and after 4 h, 4.5 h, 5 h, 5.5 h, 6 h, and 10 h (two animals each). The temporal bones and the brain tissues were collected and stained with X-gal solution as described above.

Acknowledgements We thank Drs RP Bobbin and D McCullum for their helpful advice, Brian Shin for technical assistance, and Nadine Brown for critical comments on the manuscript. This work was supported by NIH NIDCD Grant 2 P01 DC00078 (YR). TS is a scholar of the Alexander von Humboldt-Foundation.

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2 Chen P, Segil N. P27Kip1 links cell proliferation to morphogenesis in the developing organ of Corti. Development 1999; 126: 1581–1590. 3 Lo¨wenheim H et al. Gene disruption of p27Kip1 allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci USA 1999; 96: 4084–4088. 4 Raphael Y, Yagi M. Gene transfer and the inner ear. Curr Opin Otolaryngol Head Neck Surg 1998; 6: 311–315. 5 Raphael Y, Frisancho JC, Roessler BJ. Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo. Neurosci Lett 1996; 207: 137–141. 6 Weiss MA, Frisancho JC, Roessler BJ, Raphael Y. Viral-mediated gene transfer in the cochlea. Int J Dev Neurosci 1997; 15: 577–583. 7 Lalwani AK et al. Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig. Gene Therapy 1996; 3: 588–592. 8 Geschwind MD et al. Defective HSV-1 vector expressing BDNF in auditory glia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. Hum Gene Ther 1996; 7: 173–182. 9 Kiernan AE, Fekete DM. In vivo gene transfer into the embryonic inner ear using retroviral vectors. Audiol Neurootol 1997; 2: 12–24. 10 Yagi M et al. Hair cells are protected from aminoglycoside ototoxicity by adenoviral-mediated overexpression of GDNF. Hum Gene Ther 1999; 10: 813–823. 11 Staecker H, Gabaizadeh R, Federoff H, Van De Water TR. Brainderived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss. Otolaryngol Head Neck Surg 1998; 119: 7–13. 12 Lalwani AK et al. Green fluorescent protein as a reporter for gene transfer studies in the cochlea. Hear Res 1997; 114: 139–147. 13 Lalwani AK et al. Expression of adeno-associated virus integrated transgene within the mammalian vestibular organs. Am J Otol 1998; 19: 390–395. 14 Komeda M, Roessler BJ, Raphael Y. The influence of interleukin1 receptor antagonist transgene on spiral ganglion neurons. Hear Res 1999; 131: 1–10. 15 Salt AN, Thalmann R. Cochlear fluid dynamics. In: Jahn AF and Santos-Sacchi J (eds). Physiology of the Ear. Raven Press: New York, 1988, pp 341–357. 16 Woolley AL et al. Risk factors for hearing loss from meningitis in children: the Children’s Hospital experience. Arch Otolaryngol Head Neck Surg 1999; 125: 509–514. 17 Kesser BW et al. Time course of hearing loss in an animal model of pneumococcal meningitis. Otolaryngol Head Neck Surg 1999; 120: 628–637. 18 Forge A. Outer hair cell loss and supporting cell expansion following chronic gentamicin treatment. Hear Res 1985; 19: 171–182. 19 Raphael Y, Altschuler RA. Scar formation after drug-induced cochlear insult. Hear Res 1991; 51: 173–184. 20 Leonova EV, Raphael Y. Application of a platinum replica method to the study of the cytoskeleton of isolated hair cells, supporting cells and whole mounts of the organ of Corti. Hear Res 1999; 130: 137–154. 21 Salt AN, Inamura N, Thalmann R, Vora AR. Evaluation of procedures to reduce fluid flow in the fistulized guinea-pig cochlea. Acta Otolaryngol (Stockh) 1991; 111: 899–907. 22 Mondain M et al. Adenovirus-mediated in vivo gene transfer in guinea pig middle ear mucosa. Hum Gene Ther 1998; 9: 1217– 1221. 23 Davidson BL et al. A model system for in vivo gene transfer into the central nervous system using an adenoviral vector. Nat Genet 1993; 3: 219–223. 24 Sto¨ver T, Yagi M, Raphael Y. Cochlear gene transfer: round window versus cochleostomy inoculation. Hear Res 1999; 136: 124–130.

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