Introduction

Hearing and balance depend on the function of the inner ear sensory epithelium, which consists of hair cells and a number of supporting cells that provide mechanical support for the sensory cells. The development of efficient transgene delivery for the inner ear is an important step toward potential application of gene-based therapies for cochlear disorders. Recently, a number of genes implicated in inherited peripheral hearing and vestibular disorders that affect specific cell types have been described, making hearing disorders as well as progressive forms of deafness excellent targets for gene therapy¹. To deliver these genes several gene transfer vectors, including adenovirus, lentivirus, herpes simplex virus, and adeno-associated virus, were characterized both in vivo and in vitro using explants of rat inner ear sensory epithelia. While they appeared promising, each system had limitations concerning transduction efficiency, tropism, or nonspecific pathology induced by the vector^{2,3,4,5,6,7,8,9,10,11,12,13}.

Adeno-associated viruses (AAVs) are small replication-defective parvoviruses whose genomes can be easily manipulated and recombinant particles can be produced at high titers. Vectors derived from AAVs can direct long-term transgene expression and elicit a minimal immune response compared to other systems¹⁴. In vivo studies with AAV serotype 2 (AAV2) in the organ of Corti demonstrated transduction of cochlear blood vessels and neurons, but not of inner or outer hair cells¹¹. The cellular tropisms of AAV serotypes are distinct and the differences are attributed to sequence changes in the viral capsid and the differential expression of cellular proteins required for virus transduction^14,15,16. For example, AAV2 transduction is dependent upon heparan sulfate proteoglycan expression, and it can be competitively impeded by soluble heparin¹⁷. Other proteins involved in AAV2 transduction include FGFR1, integrin V5, and a novel 150-kDa protein present on the surface of permissive cells^18,19,20. In contrast, AAV4 and AAV5 are insensitive to heparin competition and exhibit distinct cell tropism in the central nervous system^14,16,21,22. Molecular characterization of the cellular factors required for transduction with these isolates demonstrates that both AAV4 and AAV5 require surface expression of 2-3 sialic acid²³. While a principle receptor has not been identified for AAV4, additional experiments have identified the platelet-derived growth factor receptors (PDGFRs) as principle receptors for efficient binding and transduction of AAV5²⁴. Other primate isolates are less well characterized but they also exhibit unique tropism. In addition, nonprimate AAVs have demonstrated gene transfer ability in distinct cell types compared with primate isolates^25,26.

Here we characterized the tropism and transduction efficiency of a novel bovine adeno-associated virus vector (BAAV)²⁶ in cultured rat inner ear epithelia and compared its transduction with that of three well-characterized, primate adeno-associated viral vectors: AAV2, AAV4, and AAV5. We show that a novel bovine vector can efficiently transduce supporting cells and hair cells of cultured inner ear epithelia. To confirm the identity of these cells we used a novel -actin–GFP fusion reporter gene, which would incorporate into the stereocilia and the apical junctional complexes of the transduced cells. This novel bovine virus was significantly more effective in transducing cells of the inner ear epithelia than other tested AAV serotypes and no pathological effects were observed.

Results

Characterization of BAAV Transduction of Hair Cells

To evaluate the tropism of this novel nonprimate bovine vector, BAAV, we incubated cultured explants of rat auditory and vestibular epithelia with BAAV expressing different reporter genes (-galactosidase, GFP, and -actin–GFP). In the preliminary experiments, we used the common reporter genes -galactosidase and GFP. The long columnar shape of hair cells and complex cellular architecture of sensory epithelia, however, made it very difficult to estimate the type and number of transduced cells based only on the diffuse cytoplasmic labeling (data not shown). To overcome this difficulty, we used -actin–GFP fusion protein as a reporter. -Actin–GFP can selectively incorporate into hair cell stereocilia^27,28 as well as into the apical junctional complexes of hair cells and supporting cells (unpublished data). This process allows a straightforward identification of transduced cells on the surface of sensory epithelia.

Previous studies have shown that for adenovirus vectors, cell tropism changes within the first few days of postnatal development with the maturation of the inner ear sensory epithelia¹³. Therefore we analyzed the tropism and transduction efficiency of BAAV in P2 and P10 inner ear explants. Analysis of fixed and counterstained P2 cultures after 8 days of incubation with 10¹⁰ DNase-resistant particles/ml (DRP/ml) of BAAV revealed transduction of both hair cells (Figs. 1A and 1E) and supporting cells (Figs. 1B–1D, 1F, and 1G). Inner and outer hair cells of the organ of Corti (Fig. 1A) as well as vestibular hair cells (Fig. 1E) showed incorporation of -actin–GFP into stereocilia beginning from their tips. This finding is identical to results obtained with gene gun plasmid delivery^27,28. We also observed incorporation of -actin–GFP into apical junctional complexes of transduced hair cells and supporting cells such as Hensen's, phalangeal, interdental, and vestibular supporting cells (Fig. 1). In all of the analyzed explants (n = 50), we did not observe any significant changes in the overall pattern of the sensory epithelia even after a prolonged incubation of 8 days with BAAV. We evaluated the potential toxic effects of actin overexpression by assessing the general appearance of the epithelia such as the organization of the rows of hair cells, missing hair bundles, or enlarged supporting cells. We visualized the hair bundles, their shape, and their ordered staircase pattern as well as the cuticular plate and actin ring using high-resolution confocal microscopy (100 objective and applying digital zoom 2–5). Evaluation of hundreds of transduced cells did not reveal any apparent structural damage. Given the cellular complexity of inner ear epithelia and the lack of appropriate cellular markers, we could not accurately determine the total number of various supporting cell types in an explant. We estimated that 100% of Hensen's cells and vestibular supporting cells were transduced and approximately 40% of the phalangeal and interdental cells were transduced. Hair cells were readily quantified by scoring stereocilia bundles and comparing them to the number of rhodamine/phalloidin-stained bundles. In P2 cultures, BAAV successfully transduced 10% of inner (n = 189) and outer (n = 773) hair cells and 48% of vestibular hair cells (n = 2032). We did not observe any differences in transduction efficiency between apical, middle, and basal turns of the organ of Corti or between different patches of vestibular epithelia.

Figure 1.

Evaluation of BAAV tropism in the organ of Corti. -Actin–GFP-positive cells (green) indicate transduction. F-actin is stained with rhodamine/phalloidin (red). (A) Adjacent auditory hair cells clearly demonstrate the advantages of using -actin–GFP as a marker. The hair cell on the right is easily scored as BAAV positive and it is morphologically identical to its neighbor. Three supporting cell types are also transduced with BAAV, and -actin–GFP was localized into short interdigitating actin filaments of the apical junctional complexes at the epithelial surfa (B) phalangeal, (C) Hensen's, and (D) interdental cells. Microvilli on the luminal surface of Hensen's cells also incorporated -actin–GFP. (E) Transduced vestibular hair cell demonstrating different degrees of -actin–GFP incorporation into stereocilia tips. (F and G) Transduced supporting cells between (*) vestibular hair cells localize -actin–GFP into the apical junctional complexes at the epithelial surface. (Bar, 5 m.). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Full figure and legend (390K)

Qualitative analysis of P10 cultures incubated with BAAV revealed a similar transduction profile of hair cells and supporting cells as observed in the P2 cultures. On the other hand, the overall yield of transduced vestibular hair cells in the older cultures was significantly lower (P < 0.05) than in P2 explants (17% in P10, n = 1549, and 48%, n = 2032, in P2 explants). Unfortunately, auditory hair cells progressively degenerate in cultures older than 15 days (unpublished data). Thus, we were unable to estimate the number of transduced inner or outer hair cells for these older cultures. Furthermore, the transduction in P2 cultures was dose-dependent; increasing titer of BAAV vector resulted in a significant increase in transduction of hair cells (Fig. 2). We observed the greatest improvement in transduction yield in vestibular hair cells, of which almost 50% were transduced after 8 days. However, the 100-fold increase in viral particles used did not correspond with an equivalent increase in transduced cells, indicating reduced transduction efficiency with increasing viral titer. To analyze if the duration of incubation with viral particles influenced the number of BAAV-transduced cells and increased the apparent transduction efficiency, we incubated P2 explants with viral particles for 5 or 8 days. We observed a significantly higher yield (P < 0.05) of transduced vestibular and outer hair cells following a longer incubation time. The number of transduced vestibular hair cells increased from 15% on day 5 to 48% on day 8 (Figs. 3A and 3B). Transduction of outer hair cells also increased 4-fold after 8 days but no significant increase of inner hair cell transduction was observed (data not shown).

Figure 2.

Comparison of transduction. Cells were transduced with increasing viral titers (10⁹ to 10¹¹ DRP/ml) of BAAV and analyzed after 8 days. Transduction increased significantly for outer hair cells (OHC; n = 5 and 13, respectively; single-factor ANOVA, P = 0.01699) and vestibular hair cells (VHC; n = 6 and 10, respectively; single-factor ANOVA, P = 0.000168), with a 100-fold increase in viral titer after 8 days of infection. The differences in transduction yield of inner hair cells (IHC) were not significant (n = 5 and 1, respectively; single-factor ANOVA, P = 0.23987). (Error bars represent SD.)

Full figure and legend (55K)

Figure 3.

Transduction is continuous and time-dependent. (A) Confocal image of vestibular epithelia after 5 days of BAAV infection. The positively transduced hair cells are easily scored even though the yield is sparse. (B) Confocal image of vestibular epithelia after 8 days of BAAV transduction demonstrating that almost half of the hair cells are transduced. There is a statistically significant increase (n = 7 and 10 frames, respectively, from at least three explants, P > 0.01) in the transduced cells after an additional 3 days of incubation. (C) Gene gun-mediated -actin–GFP incorporation in stereocilia after 8 h. The yield of transfected hair cells is one or two per vestibular epithelia explant and hair bundles are frequently damaged. (D) The initiation of -actin–GFP incorporation is asynchronous in BAAV-transduced epithelia. The extent of actin incorporation into stereocilia varies from just the tips of a hair bundle labeled (arrows) to the entire bundle (arrowheads). (Bar, 10 m.)

Full figure and legend (274K)

The observation that the yield of transduced cells increased over time is consistent with similar observations in vivo¹⁴. Interestingly, closer examination of the hair bundles revealed that many of the transduced hair cells incorporated -actin–GFP only at the stereocilia tips (Fig. 3D). Previous studies showed that -actin–GFP was progressively incorporated into stereocilia starting from the tips as early as 4–6 h after transfection using a gene gun (Fig. 3D). Within 48 or 72 h, the entire stereocilia bundle was labeled in auditory and vestibular hair cells, respectively^27,28. The presence of hair cells showing incorporation of -actin–GFP at the stereocilia tips after 8 days of incubation with virus may indicate that the onset of viral transduction can occur throughout the course of experimental exposure (Fig. 3D).

The substantial differences in transduction efficiency between supporting cells and hair cells prompted us to evaluate the ability of BAAV to transduce other polarized epithelia. We extended the panel of epithelial cell lines previously characterized by testing MDCK (dog kidney epithelial cell line) and Caco-2 (human adenocarcinoma intestinal epithelial cell line) cells because of their overall similarity to inner ear sensory epithelia. Confluent cultures of MDCK and Caco-2 were incubated with BAAV expressing -actin–GFP at a concentration of 10¹⁰ DRP/ml for 8 days. Surprisingly, we did not observe any transduction in MDCK cell cultures even after 8 days of infection; however, about 20% of Caco-2 cells showed -actin–GFP expression (see Supplemental Fig. 1).

Comparison of transduction with different serotypes of AAV

Previous studies concluded that AAV2 could transduce cells in the inner ear of guinea pigs^7,10. Therefore, we compared tropism and transduction efficiency of BAAV in inner ear epithelia with the other well-characterized serotypes AAV2, AAV4, and AAV5. We incubated cultured explants of rat auditory and vestibular epithelia (P2) with AAV2, -4, or -5 or BAAV expressing -actin–GFP at a concentration of 10¹⁰ DRP/ml for 8 days. Confocal analysis of fixed and counterstained samples revealed that overall BAAV was the most effective vector for transduction of hair cells and supporting cells in cultured inner ear sensory epithelia. With BAAV, we counted transduction of 48% of vestibular hair cells, 16% of auditory hair cells, 100% of Hensen's cells, and 40% of phalangeal cells. On the other hand, cultures incubated with AAV2 showed transduction in 4% of inner hair cells and AAV5 transduced 1% of the vestibular hair cells. We did not observe transduction of supporting cells with either AAV2 or AAV5 or transduction with AAV4 (Fig. 4).

Figure 4.

Comparison of the transduction efficiency of BAAV with that of three primate AAVs. Cochlear and vestibular explants were incubated with virus for 8 days and transduction yields were measured per sample frame. (A) BAAV transduced auditory and vestibular hair cells. In contrast, AAV2, -4, and -5 were surprisingly ineffective. Arrowheads indicate transduced hair cells. (Bar, 20 m.) (B) In general, the primate-derived adeno-associated viruses were comparatively ineffective as vectors for hair cells.

Full figure and legend (261K)

Discussion

BAAV effectively transduces hair cells as well as supporting cells in the inner ear epithelia

Explants harvested from the developing inner ear have proved to be a useful system for studying gene expression in this tissue²⁹. In this article we demonstrated that a novel, bovine adeno-associated virus vector can efficiently transduce developing and mature hair cells as well as supporting cells of inner ear explants. Our observation that morphologically mature vestibular hair cells of P10 explants can be transduced with BAAV is encouraging and further suggests that gene transfer studies using BAAV may successfully transduce hair cells of adult animals. Several studies using AAV2 serotypes in adult animals have demonstrated transduction of hair cells and supporting cells in the inner ear^7,9,10,11. However, we found that AAV2 serotypes were much less effective at transducing hair cells than BAAV. While the slow kinetics of transduction associated with AAV2 vectors may account for the low AAV2 transduction in these short-term in vitro experiments compared to BAAV, species differences between rat and guinea pig may also explain these observations. In an accompanying paper, Stone et al. demonstrate that efficient AAV2 transduction of hair cells requires the use of strong transcription elements³⁰. In agreement with our findings, even the use of strong promoter elements does not result in efficient transduction activity with AAV5 vectors in the inner ear³⁰.

-Actin–GFP Is an Optimal Reporter Gene for Inner Ear Epithelia

-Actin–GFP used in these studies as a reporter gene for analysis of tropism and transduction of viral vectors allowed for the easy identification of transduced and nontransduced cells in whole-mount preparations of sensory epithelia based on labeling of the hair bundle. In addition, localization of -actin–GFP into the apical junctional complexes of hair cells and supporting cells highlighted the cell borders in these complex mosaics, simplifying counting of the transduced cells. Finally, the ability to follow turnover of stereocilia actin in cells expressing -actin–GFP allows for the determination of initiation of transgene expression.

BAAV—a Novel Mammalian AAV Serotype

Recombinant BAAV appears to have several attributes that make it an attractive vector for gene transfer, including unique serological identity, cell tropism, and efficient gene transfer in vivo²⁶. However, we currently do not know what capsid interactions are required for efficient transduction with this serotype. BAAV is the third dependovirus of nonprimate origin to be cloned and sequenced. The high homology between BAAV and AAV5 rep along with the biochemically distinct mechanisms of replication for these two viruses compared to other mammalian AAVs suggests that BAAV and AAV5 might form a distinct group within the dependovirus genus. However, the capsid of BAAV is most similar to that of AAV4, with the divergent regions clustered mainly on the exposed surface loops that comprise the three axes of symmetry. This region is critical for AAV2 transduction^26,31,32 and may help explain the distinct tropism of BAAV vectors.

Molecular basis of specificity of AAV serotypes

In contrast to BAAV, AAV2 and AAV5 were less effective in transducing hair cells. Our in vitro results with AAV2 are consistent with the in vivo studies using AAV2 since less then 2% of the hair cells were transduced¹¹. Efficient AAV2 transduction requires expression of heparan sulfate proteoglycan on the target cell surface. Heparan sulfate cytochemistry indicated that hair cells do not express this glycoprotein residue on their apical cell surface¹². Efficient transduction with AAV5 vectors requires the expression of two or three sialic acid residues and PDGFR or PDGFR^23,24. Sialic acid residues are very sparsely distributed on the apical surface of hair cells and PDGFR receptors are localized only to the lateral wall of vestibular hair cells and not in the apical surface, limiting their utility for transduction^33,34.

Efficient gene transfer to the cochlea offers both the potential of new therapies for deafness and the ability to study specific genes and their function. The nearly 100% gene transfer in supporting cells may prove useful clinically because many genetic hearing loss diseases are caused by mutations that affect the supporting cell integrity³⁵. Proteins from the connexin family form gap junctions between supporting cells and disruption of these junctions leads to dysfunction of sensory epithelia³⁶. Recent studies indicate that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo^37,38. Most importantly, the availability of a vector that efficiently transduces hair cells in vivo would advance our ability to characterize the structure and function of the inner ear. The combination of efficiency and lack of adverse effects makes BAAV an exciting new vector choice for gene transfer to the sensory and nonsensory cells of the inner ear.

Materials and methods

Reagents

Rhodamine/phalloidin and ProLong anti-fade mounting media were from Molecular Probes (Eugene, OR, USA). Cell Tak was from BD Biosciences (Palo Alto, CA, USA). DMEM F-12, L-15 medium, fetal bovine serum, and ampicillin were from GIBCO (Carlsbad, CA, USA).

AAV vector construction, preparation, and quantification

The construction of the -galactosidase and GFP expression plasmids has been described previously^26,16. The AAV2 vector plasmid containing the AAV2 inverted terminal repeats (ITRs) flanking the CMV–-actin–GFP fusion expression cassette was constructed by subcloning of the CMV promoter -actin–GFP cassette from the -actin–GFP plasmid (BD Biosciences) into the AAV2 RSV–GFP expression plasmid and replacement of the RSV–GFP cassette with the CMV -actin–GFP. The AAV5 -actin–GFP fusion expression plasmid was produced in the same manner; however, the CMV -actin–GFP cassette was cloned into the AAV5 RSV–GFP plasmid, which contained the AAV5 ITRs.

Recombinant AAV particles were produced by triple transfection of 293T cells with an AAV helper plasmid (expressing the AAV Rep and Cap genes), an AAV vector plasmid (containing the reporter gene flanked by either type 2 ITRs (AAV2, AAV4) or type 5 ITRs (AAV5, BAAV), and the Ad helper plasmid pAd12²⁶. Recombinant vectors were purified by fractionation with CsCl-gradient centrifugation. DNase resistant genome copy titers of the vector preparations were determined by quantitative real-time PCR using the TaqMan system (Applied Biosystems) with probes specific to the CMV promoter. Viruses in CsCl were dialyzed for 24 h using 0.5 ml Slide-A-Lyzer (Pierce) in 100 ml of serum-free medium with changing of the medium three or four times.

Organotypic cultures of rat sensory epithelia

Organotypic cultures of Sprague–Dawley rat (Taconic) organ of Corti and vestibular sensory epithelia were prepared according to a published method³⁹. P0–1 rat pups were anesthetized using CO₂ according to NIH guidelines and the skin was cleaned thoroughly with 70% ethanol. After decapitation, both temporal bones were isolated and placed into L-15 medium under sterile conditions. After each otic capsule was opened, the stria vascularis, spiral ganglion, Reisner's membrane, and tectorial membrane were removed from all turns of the cochlea. The isolated organ of Corti was divided for culturing and pieces of apical, middle, and basal turns from one organ of Corti were placed in a single culture dish. Subsequently, the maculae of the sacculae and utriculae along with cristae ampullaris were finely dissected and the otoconial membrane was removed. All vestibular epithelia from one temporal bone were placed in a single culture dish. Each sample of the sensory epithelia was mounted on a Cell Tak-coated coverslip on the bottom of modified culture dish. Cultures were maintained at 37°C and 5% CO₂ in DMEM F-12 supplemented with 7% fetal bovine serum containing 1.5 g/ml ampicillin.

Viral transduction and histochemistry

Cultured explants of auditory and vestibular sensory epithelia were transduced with AAV2, AAV4, AAV5, and BAAV viral vectors using -actin–GFP as a reporter gene (BD Biosciences). Briefly, cultures were incubated with virus for 24 h in 200 l of medium. Additional medium was then added up to 1 ml and maintained for the duration of the experiment. For immunohistochemistry, cultures were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature and permeabilized for 30 min with 0.5% Triton X-100 in PBS, and the actin filaments were counterstained with rhodamine/phalloidin (0.2 U/200 l) for 30 min. Stained explants were removed from the culture dish and mounted using ProLong anti-fade medium. Fluorescence images were obtained with a Zeiss LSM 510 confocal microscope using a 40, 1.3 numerical aperture (NA), and 100 1.4 NA objectives. Image acquisition and post-acquisition analyses were performed using Adobe PhotoShop.

Statistical analyses

To quantify the transduction efficiency we took several overlapping frames from each piece of inner ear epithelia with a 40 objective and digital zoom (1.5) to see hair bundles clearly and obtain a wide field of view (usually three to seven frames to cover one explant). For each analysis, frames were scored for GFP-positive cells, and total number of cells was determined by scoring the rhodamine/phalloidin-positive cells. At least three ears from three different animals were used for each experiment. Single-factor ANOVA and Student's t test analyses were performed using Microsoft Excel.

References

1. Frolenkov, G. I., Belyantseva, I. A., Friedman, T. B. and Griffith, A. J. (2004). Genetic insights into the morphogenesis of inner ear hair cells. Nat. Rev. Genet. 5: 489–498. | Article | PubMed | ISI | ChemPort |
2. Holt, J. R., (2002). Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear. Audiol. Neurootol. 7: 157–160. | Article | PubMed | ChemPort |
3. 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 |
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., et al. (2002). Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea. Mol. Ther. 6: 12–18. | Article | PubMed | ChemPort |
7. 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 |
8. Lalwani, A. K., Walsh, B. J., Carvalho, G. J., Muzyczka, N. and Mhatre, A. N. (1998). Expression of adeno-associated virus integrated transgene within the mammalian vestibular organs. Am. J. Otol. 19: 390–395. | PubMed | 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. 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 |
11. 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 |
12. 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 |
13. Kanzaki, S., Ogawa, K., Camper, S. A. and Raphael, Y. (2002). Transgene expression in neonatal mouse inner ear explants mediated by first and advanced generation adenovirus vectors. Hear. Res. 169: 112–120. | Article | PubMed | ChemPort |
14. 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 |
15. Grimm, D. and Kay, M. A. (2003). From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr. Gene Ther. 3: 281–304. | Article | PubMed | ChemPort |
16. 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 |
17. Summerford, C. and Samulski, R. J. (1998). Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72: 1438–1445. | PubMed | ISI | ChemPort |
18. Qing, K., Mah, C., Hansen, J., Zhou, S., Dwarki, V. and Srivastava, A. (1999). Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat. Med. 5: 71–77. | Article | PubMed | ISI | ChemPort |
19. Mizukami, H., Young, N. S. and Brown, K. E. (1996). Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein. Virology 217: 124–130. | Article | PubMed | ISI | ChemPort |
20. Summerford, C., Bartlett, J. S. and Samulski, R. J. (1999). AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat. Med. 5: 78–82. | Article | PubMed | ISI | ChemPort |
21. 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 |
22. Davidson, B. L. and Chiorini, J. A. (2003). Recombinant adeno-associated viral vector types 4 and 5: preparation and application for CNS gene transfer. Methods Mol. Med. 76: 269–285. | PubMed | ChemPort |
23. Kaludov, N., Brown, K. E., Walters, R. W., Zabner, J. and Chiorini, J. A. (2001). Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J. Virol. 75: 6884–6893. | Article | PubMed | ISI | ChemPort |
24. Di Pasquale, G., et al. (2003). Identification of PDGFR as a receptor for AAV-5 transduction. Nat. Med. 9: 1306–1312. | Article |
25. Bossis, I. and Chiorini, J. A. (2003). Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles. J. Virol. 77: 6799–6810. | Article | PubMed | ChemPort |
26. Schmidt, M., Katano, H., Bossis, I. and Chiorini, J. A. (2004). Cloning and characterization of a bovine adeno-associated virus. J. Virol. 78: 6509–6516. | Article | PubMed | ChemPort |
27. Schneider, M. E., Belyantseva, I. A., Azevedo, R. B. and Kachar, B. (2002). Rapid renewal of auditory hair bundles. Nature 418: 837–838. | Article | PubMed | ISI | ChemPort |
28. Rzadzinska, A. K., Schneider, M. E., Davies, C., Riordan, G. P. and Kachar, B. (2004). An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal. J. Cell Biol. 164: 887–897. | Article | PubMed | ISI | ChemPort |
29. He, D. Z., Zheng, J. L. and Dallos, P. (2001). Development of acetylcholine receptors in cultured outer hair cells. Hear. Res. 162: 113–125. | Article | PubMed | ChemPort |
30. Stone, I. M., Lurie, D. I., Kelley, M. W. and Poulsen, D. J. (2005). Adeno-associated virus mediated gene transfer to hair cells and support cells of the murine cochlea. Mol. Ther. 11: 843–848. | Article | PubMed | ChemPort |
31. Kern, A., et al. (2003). Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J. Virol. 77: 11072–11081. | Article | PubMed | ISI | ChemPort |
32. Opie, S. R., Warrington, K. H. Jr., Agbandje-McKenna, M., Zolotukhin, S. and Muzyczka, N. (2003). Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J. Virol. 77: 6995–7006. | Article | PubMed | ISI | ChemPort |
33. Suzuki, H., Katori, Y., Ikeda, K. and Takasaka, T. (1995). Carbohydrate distribution in the living utricular macula of the guinea pig detected by lectins. Hear. Res. 87: 32–40. | Article | PubMed | ChemPort |
34. Saffer, L. D., Gu, R. and Corwin, J. T. (1996). An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles. Hear. Res. 94: 14–23. | Article | PubMed | ChemPort |
35. Tucker, J. B., Mackie, J. B., Bussoli, T. J. and Steel, K. P. (1999). Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: response to loss of cell partners in the Bronx waltzer mouse. J. Neurocytol. 28: 1017–1034. | Article | PubMed | ChemPort |
36. Cohen-Salmon, M., et al. (2002). Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr. Biol. 12: 1106–1111. | PubMed | ChemPort |
37. Zheng, J. L. and Gao, W. Q. (2000). Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3: 580–586. | Article | PubMed | ISI | ChemPort |
38. Kawamoto, K., Ishimoto, S., Minoda, R., Brough, D. E. and Raphael, Y. (2003). Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo. J. Neurosci. 23: 4395–4400. | PubMed | ChemPort |
39. Sobkowicz, H. M., Loftus, J. M. and Slapnick, S. M. (1993). Tissue culture of the organ of Corti. Acta Otolaryngol. Suppl. 502: 3–36. | PubMed | ChemPort |

Appendices

Appendix A

Supplementary data

Supplementary data for this article may be found on ScienceDirect.