Introduction

The total number of hair cells in the cochlea is finite. They are not renewed and there is very little (if any) redundancy in this population. The irreversible loss of cochlear hair cells is presumed to be a fundamental cause of permanent sensorineural hearing loss. Gene transfer into hair cells presents numerous opportunities for protecting these cells. There is considerable interest in the development of viral vectors to deliver genes to the cochlea to counteract hearing impairment, and recent studies have focused on vectors based on adenovirus^1,2,3, herpes simplex virus^4,5,6, lentivirus⁷, and adeno-associated virus (AAV)^8,9. The patterns of vector-encoded transgene expression have been found to differ significantly among vectors. Cochlear hair cells can be efficiently transduced with adenovirus vectors^10,11,12. However, these vectors were found to provoke a strong immune response that could damage recipient cells and compromise cochlear function^10,13,14; they are also incapable of mediating prolonged transgene expression^15,16. Although AAV vectors might overcome these problems, the transduction of hair cells by AAV2-derived vectors is controversial^8,10,17. To our knowledge, other AAV serotypes have not yet been tested as cochlear gene transfer vectors in vitro or in vivo. AAV vectors are of interest in the context of gene therapy because they mediate efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity.

In this study, we assessed the utility of seven AAV serotypes as vectors with the chicken -actin promoter associated with a cytomegalovirus immediate-early enhancer (CAG)-driven enhanced green fluorescent protein (EGFP) gene¹⁸ in the murine cochlea. Vectors were introduced by microinjection through the round window membrane¹⁹. As a result, we determined that the specific and efficient gene transduction of inner hair cells could be achieved by using AAV type 3 vectors.

Results

Expression profile of EGFP in the cochlea

Several cell types line the cochlear duct and support the hair cells (Fig. 1A). We carefully made a small opening in the tympanic bulla and injected vectors derived from the AAV1–4, 7, and 8 pseudotypes into the cochlea of two strains of mice (C57BL/6J and ICR) through the round window membrane (Fig. 1B). The mode of EGFP expression in various murine cochlear hair cells had a close similarity and was essentially equal for both strains. We determined the distribution of AAV vector-mediated EGFP expression throughout the cochlea for all serotypes tested (Table 1). A principal finding is that the inner hair cells in the organ of Corti showed clear evidence of EGFP expression with all of the AAV serotype-derived vectors except for the AAV4 vector (Fig. 2). This result indicates that most of the vectors (AAV1–5, 7, and 8) could efficiently transduce cochlear inner hair cells in vivo when slowly infused into the scala tympani. The AAV3-based vector was the most efficient and specific of the serotypes in transducing cochlear inner hair cells (Fig. 3). Transduction with 510¹⁰ genome copies (gc)/cochlea of the AAV3 vector resulted in robust transgene expression in the inner hair cells. The spiral ganglion cells showed significantly higher levels of fluorescence per unit area with the AAV5-based vector (Fig. 2n), and the spiral ligament cells were transduced prominently with the AAV1 and AAV7 vectors (Figs. 2d and 2r). Histological sections of cochleae injected with the AAV4 vector identified EGFP-positive cells predominantly in connective tissue within the mesothelial cells beneath the organ of Corti and in mesenchymal cells lining the perilymphatic fluid spaces (Figs. 2j and 2l). Furthermore, we detected intense expression with the AAV5- and AAV8-based vectors in the inner sulcus cells and in Claudius' cells (Figs. 2p and 2x). We did not detect notable levels of gene expression in the outer hair cells, supporting pillar cells, or stria vascularis cells for any serotype.

Figure 1.

(A) Schematic diagram of a cross section of the cochlea, demonstrating the scala vestibuli, scala tympani, and scala media or cochlear duct. The organ of Corti rests on the basilar membrane, with the hair cell cilia embedded in the gelatinous tectorial membrane. The outer margin of the cochlear duct contains the stria vascularis. Reproduced, by permission of the publisher, from⁴⁴. (B) Direct visualization of the round window membrane in the right ear. The upper side of the picture is the back of the mouse and the right side is the head of the animal. The stapedial artery, a branch of the internal carotid artery, transverses an open bony semicanal within the round window niche. Bar denotes 500 m, 15 original magnification.

Full figure and legend (138K)

Figure 2.

Transduction of the cochleae by AAV1-, AAV2-, AAV4-, AAV5-, AAV7-, and AAV8-based vectors. (a, c, e, g, i, k, m, o, q, s, u, and w) Light photomicrographs of cochlear cryosections. (b, d, f, h, j, l, n, p, r, t, v, and x) Fluorescence photomicrographs (green fluorescence from transgene). The spiral ligament cells were transduced prominently with the AAV1 and AAV7 vectors (d and r). Transgene expression in inner hair cells was detected with AAV1-, AAV2-, AAV5-, AAV7-, and AAV8-based vectors (b, h, n, t, and x). AAV4-based vector faintly transduced mesenchymal cells (j and l). The spiral ganglion cells showed significant levels of fluorescence with the AAV5-based vector (n). Intense fluorescence was detected with the AAV5- and AAV8-based vectors in the inner sulcus cells (p and x). Scale bars: 10, 100 m; 20, 50 m; 40, 25 m; 60, 25 m.

Full figure and legend (464K)

Figure 3.

Cochlear transduction with AAV3-CAG-EGFP. Dissected cochleae and cryosections show transgene expression in inner hair cells. (a) A light photomicrograph of the basal turn of the cochlea is shown, illustrating its laminar structure. (b) A fluorescence photomicrograph of this dissection. (c) A higher magnification view of the dissection shown in (b), illustrating a row of inner hair cells in the organ of Corti expressing EGFP. (d–i) Representative photomicrographs from three magnifications of a radial cochlear cryosection. (d) Light photomicrography of an intact cochlear duct. Fluorescence photomicrography of this duct is shown in (e). (h and i) A higher magnification of (e), illustrating EGFP expression within inner hair cells. Cryosections show transgene expression in the inner hair cells (arrows). Scale bars: 4, 250 m; 10, 100 m; 20, 50 m; 40, 25 m; 60, 25 m.

Full figure and legend (118K)

Table 1 - Expression of transgene in the mouse cochlea with vectors derived from the AAV1–4, 7, and 8 pseudotypes.

Full table

Long-term expression of EGFP

We examined cochlear expression of the EGFP transgene in animals sacrificed at 1–12 weeks. Expression persisted in cochlear tissues for up to 3 months after infusion, while the extent of expression peaked at 2 weeks.

Transgene activity

We determined the percentage of inner hair cells transduced with the AAV3 vector. The mid- to high-frequency regions of the cochlea were efficiently transduced, as shown in Fig. 3. Almost all of the inner hair cells in the basal and middle cochlear regions were transduced with the AAV3 vector (Fig. 4). Transgene expression was not detected in the hair cells of the apical turn of the cochlea. The predominant expression in the middle and basal cochlear turns is reasonable, as the virus was slowly infused into the scala tympani adjacent to the most basal turn of the cochlea. The percentage of transduced inner hair cells from the basal (high frequencies) to the apical (low frequencies) cochlear regions is shown in Fig. 4.

Figure 4.

EGFP expression profile of inner hair cells transduced with AAV3, as shown for a cross section subdivided into 12 segments ranging from the basal (high frequencies) to the apical (low frequencies) cochlear regions.

Full figure and legend (47K)

Cytotoxicity

We detected no deleterious effects on the viability of transduced cells. We compared evoked auditory brain-stem response (ABR) threshold levels before and after injection, using a two-way repeated measure of the analysis of variance. There was no significant loss in ABR and hence no change in cochlear function for up to 10 days following vector infusion (Figs. 5A and 5B). In addition, the cellular and tissue architecture of experimental cochleae remained intact. There was no evidence of endolymphatic hydrops after AAV vector injection in any of the animals. We observed no significant destruction of the inner or outer hair cells (Fig. 5C).

Figure 5.

(A) ABR threshold (meanSD) at each frequency tested preoperatively (pre) versus postoperatively (post). (B) Example of ABR waveforms in C57BL/6J at various stimuli (16 kHz; 108 dB, 78 dB, 48 dB, 28 dB, and 23 dB). ABR were tested in the transduced ear prior to viral injection and 10 days after injection. Wave I was measured to analyze the activity of the cochlea. (C) F-actin staining showing that no outer hair cells were lost from inoculated cochleae. Original magnification 40; scale bar, 25 m.

Full figure and legend (228K)

Discussion

In the present study, we assessed the utility of vectors derived from seven AAV serotypes for gene delivery into the cochlea. Our results showed that the AAV3 vector was the most efficient and specific in transducing cochlear inner hair cells, although these cells could also be transduced with AAV1, 2, 5, 7, and 8 vectors. The transduction efficiency of the spiral ganglion by the AAV5 vector was particularly high, followed by that of the AAV1, AAV2, and AAV7 vectors. The efficient and specific transduction of inner hair cells with the AAV3 vector suggests that it recognizes a unique host range with a distinct cellular receptor. Transduction efficiency is dependent on initial viral binding (a property of the viral capsid), entry, and various postentry processes such as intracellular trafficking and second-strand synthesis^20,21,22. The genome size of AAV vectors has also been demonstrated to affect transduction efficiency²³. Comparisons of the serotypes have indicated that heterogeneity in the capsid-encoding regions and a differential ability to transduce cells may be associated with different receptor and co-receptor requirements for cell entry²⁴. However, the receptors and co-receptors of AAV3 have not yet been clearly identified.

In the current study, we found that cochlear inner hair cells could be transduced with six AAV serotypes, although Lalwini et al.⁸ reported that outer hair cells could be transduced with a low titer (110⁶ viral particles/ml) of AAV2 in vivo. After injecting the AAV2 vector, we found that the spiral ganglion neurons, the inner hair cells, and the cells in the spiral ligament were all transduced. This transduction pattern differs from that reported in previous studies^8,10,17, and this discrepancy might be due to the different delivery methods and dissimilar promoters. Although the CAG promoter directs higher expression than do the cytomegalovirus (CMV) and EF-1 promoters²⁵, each promoter drives reporter gene expression in different cell types^26,27.

Cell-specific or -selective infectivity of the viral vectors suggests the presence of various factors to introduce the distinct expression patterns of the transgenes. Spiral ganglion neurons and glial cells can be transduced with a lentivirus–GFP construct in vitro but not in vivo⁷. The differential transducibility under in vivo and in vitro conditions reflects a high degree of structural isolation of the spiral ganglion and other cell types—such as the cells on the periphery of the endolymph—from the perilymph into which the viral vector was introduced. The strict separation of the endolymph from the perilymph is maintained by tight junctions that line the boundary between these fluid chambers. The size of the viral particle may contribute to the observed variability in transgene expression promoted by different vectors. The diameters of adenovirus and retrovirus (including lentivirus) particles are approximately 75 nm and greater than 100 nm, respectively, while the diameters of AAV vectors are typically 11–22 nm^28,29. Thus, the larger size of lentiviruses and adenoviruses may limit their subsequent dissemination from the perilymph into the endolymph. The variable patterns of adenovirus- and lentivirus-mediated gene expression seen with different methods of inoculation may be due to the inoculation route, the volume and number of viral particles, differences in viral preparation, or differences in the method of transgene detection. The introduction of adenovirus vectors by cochleostomy or with an osmotic pump via the round window leads to a more efficient transduction of cochlear hair cells^30,31,32. The apical domain (apical membrane and stereocilia) of cells in the sensory epithelium (hair cells and supporting cells) is bathed in endolymph, while the basal–lateral domain is immersed in perilymph. Access of the viral vectors to the endolymphatic space by cochleostomy may facilitate the transduction of hair cells and supporting cells. However, although the cochleostomy procedure has been tested, inoculation into the membranous labyrinth could not be confirmed³². In the present study, AAV vectors were found to infect cochlear hair cells easily in vivo, via round window injection.

Gene transfer into the cochlea through the round window membrane is ideal, because this procedure requires simple surgery without cochlear trauma¹⁹. Another critical factor in assessing the utility of a gene transfer vector is safety. Factors determining safety include the toxicity of the gene transfer agent itself, the provocation of immune responses, the generation of replication-competent virus, and the risk of creating genetically modified cells by insertional mutagenesis. The cells and tissues within the AAV-EGFP-perfused cochleae were free from inflammation and were generally intact. No pathological changes were observed in the organ of Corti, stria vascularis, or spiral ganglion cells. The long-term expression of EGFP within the cochlear tissues is consistent with data obtained from other animal models and different organ systems^9,33. Since EGFP is known to introduce cellular toxicity, vectors expressing physiologically therapeutic proteins would achieve longer transduction periods than EGFP. Gene transfer into the inner hair cells presents numerous opportunities for auditory neuroscience. Potential applications include the localization of proteins by expression of tagged constructs, the generation of dominant-negative or antisense knockouts of endogenous proteins, the rescue of mutant phenotypes to identify disease genes, and perhaps even the treatment of auditory disorders. Advances in the molecular basis of auditory diseases have allowed the identification of a number of genetic disorders such as presbycusis, acoustic trauma, and ototoxicity. The development of gene therapy now allows us to evaluate the effects of transferring therapeutic genes into the inner ear by several different strategies. The expression of marker genes in the inner ear tissue has been demonstrated. Further studies will improve our understanding of cochlear function as well as provide for the development of novel therapies for a wide variety of inner ear diseases. Intracochlear gene transfer using AAV vectors has been established as a viable experimental proposition. Future study will include the transfer of functioning genes in vivo and the development of alternative vectors. While clinical application may be some way off, it is vital that gene delivery techniques are optimized in anticipation of future need.

In conclusion, the data presented in this paper demonstrate successful gene transfer into several types of cochlear cells in vivo with AAV-based vectors. Interestingly, the AAV3 vector promoted inner hair cell-specific transduction. These findings are of value for further molecular studies of the cochlear inner hair cells and for gene replacement strategies to correct hereditary hearing loss due to specific monogenic mutations affecting cochlear inner hair cells.

Materials and methods

Construction and preparation of proviral plasmids

The AAV vector proviral plasmid pAAV2-LacZ harbors an Escherichia coli -galactosidase expression cassette with the CMV promoter, the first intron of the human growth hormone gene, and the SV40 early polyadenylation sequence, which are flanked by inverted terminal repeats (ITRs)³⁴. The LacZ expression cassette of pAAV2-LacZ was ligated to NotI-excised pAAV5-RNL³⁵ to form the proviral plasmid pAAV5-LacZ. The pAAV2-CAG-EGFP-WPRE construct consists of the EGFP gene under the control of the CAG promoter (the chicken -actin promoter associated with the cytomegalovirus immediate-early enhancer) and WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) flanked by ITRs. The WPRE cassette augments the stability of transgene mRNA³⁶ and increases EGFP expression levels, thereby ensuring long-term transgene expression. A BamHI–XbaI fragment containing the EGFP cDNA excised from pEGFP-1 and a HindIII fragment containing the WPRE sequence excised from pBS II SK⁺WPRE-B11 (a gift from Dr. J. Donello) was ligated to XhoI linkers and cloned into an XhoI site of pCAGGS (a gift from Dr. J.-I. Miyazaki) to create pCAG-EGFP-WPRE. The EGFP expression cassette from pCAG-EGFP-WPRE was ligated to the NotI-excised pAAV2-LacZ and pAAV5-RNL³⁵ to form the proviral plasmids pAAV2-CAG-EGFP-WPRE and pAAV5-CAG-EGFP-WPRE, respectively. The AAV-helper plasmid harbors Rep and Cap. The adenovirus helper plasmid pAdeno5 (identical to pVAE2AE4-5) encodes the entire E2A and E4 regions and the VA RNA I and II genes³⁷. Plasmids were purified with the Qiagen plasmid purification kits (Qiagen K.K., Tokyo, Japan).

Recombinant AAV vector production

Vectors derived from the AAV1–4, 7, and 8 pseudotypes were produced with the AAV packaging plasmid pAAV1RepCap (for AAV1)³⁸, pHLP19 (for AAV2), pAAV3RepCap (for AAV3)³⁹, pAAV4RepCap (for AAV4)⁴⁰, pAAV7RepCap (for AAV7)⁴¹, or pAAV8RepCap (for AAV8)⁴¹ and the AAV proviral plasmid pAAV2-LacZ or pAAV2-CAG-EGFP-WPRE. The plasmids pAAV5RepCap³⁵ and pAAV5-LacZ, or pAAV5-CAG-EGFP-WPRE, were used to produce vector with the AAV5 pseudotype⁴². Seven AAV serotype vectors were produced as previously described by the three-plasmid transfection adenovirus-free protocol³⁷. Briefly, three days before transfection, 293 cells were plated onto a 10-tray Cell Factory (Nalge Nunc International, Rochester, NY, USA; 610⁷ cells/10-tray). The cells were cotransfected with 650 g each of the proviral plasmid, the AAV vector packaging plasmid, and the adenovirus helper plasmid pAdeno5³⁴ by the calcium phosphate coprecipitation method. The medium was changed following incubation for 6–8 h at 37°C. Recombinant AAV was harvested 72 h after transfection by three freeze/thaw cycles. The crude viral lysate was purified twice on a cesium chloride two-tier centrifugation gradient as described previously²⁴. The viral stock was treated with DNase and titrated by quantitative real-time PCR with plasmid standards⁴³.

Surgical procedures and cochlear perfusions

All animal studies were performed in accordance with the guidelines issued by the committee on animal research of Jichi Medical School and approved by its ethics committee. Sixty female C57BL/6J mice (4 weeks of age; CLEA Japan, Tokyo, Japan) and 40 male ICR mice (2 months of age; Japan SLC, Shizuoka, Japan) were utilized. The mice were initially anesthetized with ketamine (50 mg/kg) and the analgesic xylazine (5 mg/kg). A postauricular approach was used to expose the tympanic bony bulla. A small opening (2 mm) in the tympanic bulla was carefully made to allow access to the round window membrane. In the tested groups, 5 l AAV vector solution (510¹⁰ gc) was microinjected into the cochlea through the round window over 10 min with a glass micropipette (40 m in diameter) fitted on a Univentor 801 syringe pump (Serial No. 170182, High Precision Instruments, Univentor Ltd., Malta)¹⁹. A small plug of muscle was used to seal the cochlea and the surgical wound was closed in layers and dressed with antibiotic ointment. Five mice of each strain received control cochlear perfusions with artificial perilymph (145 mM NaCl, 2.7 mM KCl, 2 mM MgSO₄, 1.2 mM CaCl₂, 5 mM Hepes) alone. Each AAV-EGFP serotype was injected into five mice of each strain. Another 20 C57BL/6J mice were injected with the AAV3 vector to study long-term expression.

Cochlear function assessment using ABR

To assess the physiological status of experimental ears, auditory thresholds were determined with multiple frequency and intensity tone bursts by performing ABR audiometry with Tucker–Davis Technologies and Scope v3.6.9 software (Power Lab/200; ADInstruments, Castle Hill, Australia). Tone pipes were introduced into the operated ears of the anesthetized mice, and evoked potentials were recorded using needle electrodes inserted through the skin. ABR were elicited and measured 256 times at 4, 8, 12, 16, 20, and 24 kHz frequencies with tone bursts in systematic 5-dB steps. The rise/fall times for the tone bursts were 0.1 ms rise/ms flat (cosine gate). Free-field system was used as a calibration procedure. Wave I was measured to analyze the activity from the cochlea. The lowest stimulus level that yielded a detectable ABR waveform was defined as the threshold. ABR were tested in the infused ear prior to surgery and 10 days postsurgery. Data were statistically analyzed using repeated-measures analysis of variance followed by paired Student's t test performed with StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Values of P<0.05 were considered significant.

Histology

Cochlear transgene expression patterns were determined for all AAV serotypes by visualizing EGFP expression. The animals were sacrificed 10 days after injection, and intracardiac perfusion was performed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. The cochleae were harvested and the stapes footplates were removed. For AAV3-mediated transduction, the animals (five mice for each time point) were sacrificed 1, 2, 4, 8, or 12 weeks after inoculation. Postfixation was carried out in 4% PFA for 4 h at 4°C, and decalcification was performed in 10% EDTA for 12 days at room temperature. The cochlear half-turns were microdissected and processed and the other half-turns were prepared by cryosection (10 m) to detect EGFP expression by using an Olympus IX70 (Olympus Corp., Tokyo, Japan) fluorescence microscope with a standard fluorescein isothiocyanate filter set and Studio Lite software (Olympus Corp.). Cells that exhibited fluorescence were considered positive for transgene expression. The level of expression was graded by fluorescence intensity on a four-point scale (+, ++, +++, ++++) depending on the pixel/unit area count. Hair cell counts were carried out with dissected cochlea.

References

1. Raphael, Y., Frisancho, J. C. and Roessler, B. J. (1996). Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo. Neurosci. Lett. 207: 137–141. | Article | PubMed | ChemPort |
2. Holt, J. R., et al. (1999). Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. J. Neurophysiol. 81: 1881–1888. | PubMed | ISI | ChemPort |
3. Yamasoba, T., Suzuki, M. and Kondo, K. (2002). Transgene expression in mature guinea pig cochlear cells in vitro. Neurosci. Lett. 335: 13–16. | Article | PubMed | ChemPort |
4. Derby, M. L., Sena-Esteves, M., Breakefield, X. O. and Corey, D. P. (1999). Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors. Hear. Res. 134: 1–8. | Article | PubMed | ChemPort |
5. Chen, X., Frisina, R. D., Bowers, W. J., Frisina, D. R. and Federoff, H. J. (2001). HSV amplicon-mediated neurotrophin-3 expression protects murine spiral ganglion neurons from cisplatin-induced damage. Mol. Ther. 3: 958–963. | Article | PubMed | ISI | ChemPort |
6. Bowers, W. J., Chen, X., Guo, H., Frisina, D. R., Federoff, H. J. and Frisina, R. D. (2002). Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea. Mol. Ther. 6: 12–18. | Article | PubMed | ChemPort |
7. Han, J. J., et al. (1999). Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector. Hum. Gene Ther. 10: 1867–1873. | Article | PubMed | ISI | ChemPort |
8. Lalwani, A. K., Walsh, B. J., Reilly, P. G., Muzyczka, N. and Mhatre, A. N. (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. | PubMed | ISI | ChemPort |
9. Lalwani, A., et al. (1998). Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus. Gene Ther. 5: 277–281. | Article | PubMed | ChemPort |
10. Luebke, A. E., Foster, P. K., Muller, C. D. and Peel, A. L. (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. | Article | PubMed | ChemPort |
11. Luebke, A. E., Steiger, J. D., Hodges, B. L. and Amalfitano, A. (2001). A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function. Gene Ther. 8: 789–794. | Article | PubMed | ISI | ChemPort |
12. Staecker, H., Li, D., O'Malley, B. W. Jr. and Van De Water, T. R. (2001). Gene expression in the mammalian cochlea: a study of multiple vector systems. Acta Otolaryngol. 121: 157–163. | Article | PubMed | ChemPort |
13. Dazert, S., Aletsee, C., Brors, D., Gravel, C., Sendtner, M. and Ryan, A. (2001). In vivo adenoviral transduction of the neonatal rat cochlea and middle ear. Hear. Res. 151: 30–40. | Article | PubMed | ChemPort |
14. Ishimoto, S., Kawamoto, K., Kanzaki, S. and Raphael, Y. (2002). Gene transfer into supporting cells of the organ of Corti. Hear. Res. 173: 187–197. | Article | PubMed | ChemPort |
15. Van de Water, T. R., Staecker, H., Halterman, M. W. and Federoff, H. J. (1999). Gene therapy in the inner ear: mechanisms and clinical implications. Ann. N.Y. Acad. Sci. 884: 345–360. | Article | PubMed | ChemPort |
16. Vassalli, G., Bueler, H., Dudler, J., von Segesser, L. K. and Kappenberger, L. (2003). Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors. Int. J. Cardiol. 90: 229–238. | Article | PubMed | ISI |
17. Li Duan, M., Bordet, T., Mezzina, M., Kahn, A. and Ulfendahl, M. (2002). Adenoviral and adeno-associated viral vector mediated gene transfer in the guinea pig cochlea. Neuroreport 13: 1295–1299. | PubMed |
18. Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N. and Mhatre, A. N. (1997). Green fluorescent protein as a reporter for gene transfer studies in the cochlea. Hear. Res. 114: 139–147. | Article | PubMed | ChemPort |
19. Kho, S. T., Pettis, R. M., Mhatre, A. N. and Lalwani, A. K. (2000). Cochlear microinjection and its effects upon auditory function in the guinea pig. Eur. Arch. Otorhinolaryngol. 257: 469–472. | Article | PubMed | ChemPort |
20. Handa, A., Muramatsu, S., Qiu, J., Mizukami, H. and Brown, K. E. (2000). Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors. J. Gen. Virol. 81: 2077–2084. | PubMed | ISI | ChemPort |
21. Davidson, B. L., et al. (2000). Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc. Natl. Acad. Sci. USA 97: 3428–3432. | Article | PubMed | ChemPort |
22. Zabner, J., et al. (2000). Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J. Virol. 74: 3852–3858. | Article | PubMed | ISI | ChemPort |
23. Yang, G. S., et al. (2002). Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size. J. Virol. 76: 7651–7660. | Article | PubMed | ISI | ChemPort |
24. Okada, T., et al. (2002). Adeno-associated virus vectors for gene transfer to the brain. Methods 28: 237–247. | Article | PubMed | ChemPort |
25. Xu, L., et al. (2001). CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum. Gene Ther. 12: 563–573. | Article | PubMed | ISI | ChemPort |
26. Chung, S., Andersson, T., Sonntag, K. C., Bjorklund, L., Isacson, O. and Kim, K. S. (2002). Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20: 139–145. | Article | PubMed | ChemPort |
27. Nomoto, T., et al. (2003). Distinct patterns of gene transfer to gerbil hippocampus with recombinant adeno-associated virus type 2 and 5. Neurosci. Lett. 340: 153–157. | Article | PubMed | ChemPort |
28. Dutta, S. K. (1975). Isolation and characterization of an adenovirus and isolation of its adenovirus-associated virus in cell culture from foals with respiratory tract disease. Am. J. Vet. Res. 36: 247–250. | PubMed | ChemPort |
29. Palmer, E. and Goldsmith, C. S. (1988). Ultrastructure of human retroviruses. J. Electron Microsc. Tech. 8: 3–15. | Article | PubMed | ChemPort |
30. Stover, T., Yagi, M. and Raphael, Y. (1999). Cochlear gene transfer: round window versus cochleostomy inoculation. Hear. Res. 136: 124–130. | Article | PubMed | ChemPort |
31. Stover, T., Yagi, M. and Raphael, Y. (2000). Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer: round window versus cochleostomy inoculation. Gene Ther. 7: 377–383. | Article | PubMed | ChemPort |
32. Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N. and Raphael, Y. (2001). The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice. Mol. Ther. 4: 575–585. | Article | PubMed | ChemPort |
33. Kaplitt, M. G., et al. (1994). Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8: 148–154. | Article | PubMed | ISI | ChemPort |
34. Okada, T., et al. (2001). Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production. Biochem. Biophys. Res. Commun. 288: 62–68. | Article | PubMed | ChemPort |
35. Chiorini, J. A., Kim, F., Yang, L. and Kotin, R. M. (1999). Cloning and characterization of adeno-associated virus type 5. J. Virol. 73: 1309–1319. | PubMed | ISI | ChemPort |
36. Zufferey, R., Donello, J. E., Trono, D. and Hope, T. J. (1999). Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73: 2886–2892. | PubMed | ISI | ChemPort |
37. Matsushita, T., et al. (1998). Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 5: 938–945. | Article | PubMed | ISI | ChemPort |
38. Mochizuki, S., et al. (2004). Adeno-associated virus (AAV) vector-mediated liver- and muscle-directed transgene expression using various kinds of promoters and serotypes. Gene Ther. Mol. Biol. 8: 9–18.
39. Muramatsu, S., Mizukami, H., Young, N. S. and Brown, K. E. (1996). Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3. Virology 221: 208–217. | Article | PubMed | ISI | ChemPort |
40. Chiorini, J. A., Yang, L., Liu, Y., Safer, B. and Kotin, R. M. (1997). Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles. J. Virol. 71: 6823–6833. | PubMed | ISI | ChemPort |
41. Gao, G. P., Alvira, M. R., Wang, L., Calcedo, R., Johnston, J. and Wilson, J. M. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99: 11854–11859. | Article | PubMed | ChemPort |
42. Rabinowitz, J. E., et al. (2002). Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J. Virol. 76: 791–801. | Article | PubMed | ISI | ChemPort |
43. Veldwijk, M. R., et al. (2002). Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol. Ther. 6: 272–278. | Article | PubMed | ChemPort |
44. Kikuchi, T., et al. (1995). Anat. Embryol. (Berlin) 191: 101–118. | ChemPort |

Acknowledgements

The authors thank Avigen, Inc. (Alameda, CA, USA) for providing pAAV-LacZ, pHLP19, and pAdeno; Dr. John A. Chiorini for pAAV4RepCap (identical to pSV40oriAAV4-2), pAAV5-RNL, and pAAV5RepCap (identical to 5RepCapB); and Dr. James M. Wilson for pAAV7RepCap and pAAV8RepCap. We also thank Dr. John E. Donello (Infectious Disease Laboratory, The Salk Institute for Biological Studies) for providing pBS II SK⁺WPRE-B11 and Dr. Jun-Ichi Miyazaki (Osaka University Graduate School of Medicine) for pCAGGS. The authors also thank Mr. Takeshi Hayakawa (Bio Research Center Co., Ltd.), Ms. Miyoko Mitsu, and Ms. Kiyomi Aoki for their encouragement and technical support. This study was supported in part by (1) grants from the Ministry of Health, Labor, and Welfare of Japan; (2) Grants-in-Aid for Scientific Research; (3) a grant from the 21 Century COE Program; and (4) the High-Tech Research Center Project for Private Universities matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.