Functional Expression of Exogenous Proteins in Mammalian Sensory Hair Cells Infected With Adenoviral Vectors

Jeffrey R. Holt,1,2 David C. Johns,3 Sam Wang,1,2 Zheng-Yi Chen,1 Robert J. Dunn,4 Eduardo Marban,3 and David P. Corey1,2

 1Department of Neurobiology, Harvard Medical School and Massachusetts General Hospital;  2Howard Hughes Medical Institute, Boston, Massachusetts 02114;  3Section of Molecular and Cellular Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and  4Centre for Research in Neuroscience, Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Holt, Jeffrey R., David C. Johns, Sam Wang, Zheng-Yi Chen, Robert J. Dunn, Eduardo Marban, and David P. Corey. Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta -galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 × 107 pfu/ml were sufficient for expression in 50% of the ~1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gene transfer into sensory hair cells presents numerous opportunities for auditory and vestibular neuroscience. Potential applications include localization of proteins by expression of tagged constructs, dominant-negative or antisense knockout of endogenous proteins, rescue of mutant phenotypes to identify disease genes, and perhaps even treatment of auditory or vestibular disorders.

Unfortunately, conventional gene transfer techniques such as cationic liposomes, electroporation, or a gene gun run the risk of substantial damage to the cell membrane and possibly the transduction machinery. Viral vectors, on the other hand, offer an attractive alternative for several reasons (Breakefield et al. 1998). 1) The transfer event is via receptor-mediated endocytosis or membrane fusion and thus poses little threat to cell integrity. 2) Viruses provide a highly efficient means of delivery and expression of exogenous genes in host cells. 3) Virus-mediated gene transfer has the potential for therapeutic use in vivo, whereas most of the nonviral techniques are much less effective.

The goal of this study was to screen several viral vectors for their ability to express exogenous genes in hair cells of cultured mouse auditory and vestibular organs. Although our screen was not exhaustive, we determined adenovirus to be a suitable vector for gene transfer into inner ear hair cells. Adenovirus-mediated gene transfer was selected because it satisfied the following criteria: efficient and specific infection of hair cells, ease of vector construction, ability to produce vector at high titers, and minimal toxic effects.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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Tissue preparation

Utricles and cochlea were excised from young CD-1 mice (Charles River, Wilmington, MA) between postnatal day 0 and postnatal day 3, as described by Rüsch and Eatock (1996a). The mice were killed by cervical dislocation and decapitation. Removal of the sensory epithelium was performed in MEM (GIBCO, Gaithersburg, MD) to which 10 mM HEPES (pH 7.4) was added. From the medial surface, the bony labyrinth was opened, and the epithelia were exposed. To facilitate removal of the otoconia and otolithic membrane, the tissue was treated with protease XXVII (Sigma, St. Louis, MO) at a concentration of 100 µg/ml in MEM for 20 min. After removal of the otolithic membrane, the utricles were excised, and the surrounding tissue and nerve were trimmed away. The sensory epithelia were then mounted on round glass coverslips with Cell Tak (Collaborative Biomedical Products, Bedford, MA) and placed in an incubator at 37°C and 5% CO2 for <= 10 days. The culture medium was identical to that described previously except that it contained 10% heat-inactivated horse serum (GIBCO). Viruses were added directly to the culture media for 24 h. All times were measured from the beginning of the viral infection.

Viral vectors

HERPES SIMPLEX VIRUS. A recombinant herpes simplex virus (HSV) type 1 vector, hrR3, which contained most of the viral genome (Goldstein and Weller 1988), was used at a titer of 108 transducing units/ml (tu/ml). This vector contains the lacZ gene in place of the deleted ribonucleotide reductase gene and was driven by the endogenous HSV ribonucleotide reductase promoter. As such, the vector is replication deficient in postmitotic cells. An HSV-amplicon vector (Johnston et al. 1997), in which the viral genome was deleted, was packaged with the helper virus-free system described by Fraefel et al. (1996). In brief, a cytomegalovirus (CMV)-driven enhanced-GFP expressing amplicon was cotransfected with the helper virus cosmid set into Vero 2.2 cells. Cells were harvested 2.5 days later by scraping followed by three freeze-thaw cycles and sonication. Cellular debris was pelleted at 3,000 g, and the cell lysate containing 107 tu/ml (titered on Vero 2.2 cells) was used for experiments.

RAAV. The University of Florida Vector Core provided recombinant adeno-associated virus vectors containing enhanced GFP under three promoters: CMV at 107 infectious particles/ml (ip/ml), alpha -elongation factor promoter at 1011 ip/ml (Zolotukhin et al. 1996), and neuron-specific promoter at 1010 ip/ml (Peel et al. 1997).

RADGFP. Replication-deficient (E1a/b deleted) recombinant adenovirus containing the cDNA for enhanced-GFP driven by a CMV promoter (rAdGFP) was obtained from Genzyme (Cambridge, MA) at a titer of 1011 plaque forming units/ml (pfu/ml).

RAD-GFP-KIR2.1. The coding sequence for the human Kir2.1 gene was fused in frame to eGFP in the plasmid vector pEGFP-C3 (Clontech, Palo Alto, CA) creating pGFP-Kir2.1. The adenovirus shuttle vector pAdLox (Hardy et al. 1997) was modified to replace the CMV promoter with the ecdysone inducible promoter from pIND-1 (Invitrogen, San Diego, CA) making the vector pAdEcd. The coding sequences from pGFP-Kir2.1 was cloned into the multiple cloning site of pAdEcd making the vector pAd-GFP-Kir2.1.

RADVGRXR. The plasmid pAdVgRXR was made by cloning the dual expression cassette from pVgRXR (Invitrogen) into pAdLox. This vector constitutively expresses a modified ecdysone receptor and the retinoid X receptor. Recombinant adenovirus vectors were generated by co-transfecting CRE8 cells with 2.1 µg of purified Psi 5 viral DNA and 2.1 µg of purified shuttle vector DNA with Lipofectamine Plus (Life Technologies). Cells were incubated 5-9 days until cytopathic effects (CPEs) were observed and then freeze-thawed. Cellular debris was removed by centrifugation, and 2 ml of the supernatant was added to 90% confluent CRE8 cells and returned to the incubator until CPEs were observed. This procedure was repeated three to four times. Viruses were expanded and purified as previously described (Johns et al. 1995). Titers were determined by plaque assays. Expression was induced by adding 4 µM muristerone A (Invitrogen). All virus stocks were aliquoted and stored at -80° C.

Microscopy

Live utricle cultures were imaged with an Axioskop FS (Zeiss, Germany) fitted for differential interference contrast (DIC) and fluorescence microscopy. A FITC long-pass filter set (No. 41012, Chroma Technology, Brattleboro, VT) was used to observe GFP fluorescence. Images were acquired with an integrating charge-coupled device (CCD) camera (C2400, Hamamatsu Photonics, Japan) and analyzed with Photoshop 4.0 (Adobe Systems, Mountain View, CA).

Fixed epithelia were imaged with a BioRad MRC 1000 confocal microscope (BioRad Laboratories, Hercules, CA). These cultures were fixed in 4% formaldehyde for 20 min, rinsed in PBS (pH 7.4), and permeablized with 0.5% Triton X-100 in PBS for 20 min. To visualize the hair bundles, the tissue was stained with 100 nM rhodamine-conjugated phalloidin for 1 h (Molecular Probes, Eugene, OR). Then the tissue was rinsed with PBS and mounted with BioRad mounting medium which contained p-phenylenediamine to prevent photobleaching. To estimate the number of hair cells in the sensory epithelium we counted the number of hair bundles. This method may have resulted in a slight under estimation of the total number of hair cells present as some cells may have lost their bundles during preparation of the tissue.

Recording

We recorded voltage-dependent and transduction currents, as previously described (Holt et al. 1997, 1998). Briefly, cultures were bathed in extracellular solution that contained (in mM) 144 NaCl, 0.7 NaH2PO4, 7 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 D-glucose, and 10 HEPES-NaOH, vitamins, and amino acids as in MEM, pH 7.4. Recording pipettes had resistances between 3 and 5 MOmega and contained (in mM) 130 KCL, 0.1 CaCl2, 5 EGTA-KOH, 3.5 MgCl2, 2.5 MgATP, and 5 HEPES-KOH, pH 7.4. Currents were recorded at room temperature (22-24°C) with an Axopatch 200B (Axon Instruments, Foster City, CA), filtered at 2 kHz with an 8-pole bessel filter (Frequency Devices, Haverhill, MA), digitized at >4 kHz with a 12-bit acquisition board and pClamp6.0 (Axon Instruments) and stored on disk for off-line analysis with Origin 5.0 (MicroCal Software, Northampton, MA). Data are presented as means ± SD.

Stimulation

The mechanical stimulus was a fluid jet delivered from a glass micropipette and controlled by a fast pressure-clamp system (Denk and Webb 1992; McBride and Hamill 1995), as described previously (Holt et al. 1997, 1998). Stimulus pipettes were pulled to a tip diameter of ~10 µm, filled with standard extracellular solution, and positioned ~50 µm from the hair bundle. Stimuli were step and sinusoidal bundle deflections controlled by pClamp 6.0 software. Hair bundle deflections were monitored with a C2400 CCD camera (Hamamatsu, Japan) and recorded onto videotape. Deflections were measured off-line directly from the video image.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Screen of viral vectors

We tested three types of viral vectors for their ability to transfer reporter genes into the hair cells of organotypic cultures of the mouse utricle. Cultures exposed to HSV-amplicon vectors at titers up to 5 × 107 tu/ml for 24 h were checked daily for expression of GFP. Between days 1 and 7 postinfection, ~20% of fibroblasts expressed GFP as well as a few glial cells. However, no hair cells appeared fluorescent throughout the duration of the experiment. We did not observe expression of either of the reporter genes, GFP or lacZ, in hair cells with any of the HSV vectors we tested, which included hrR3 and amplicon vectors with titers that ranged between 106 tu/ml and 5 × 108 tu/ml.

Adeno-associated virus was introduced into the culture medium at titers of <= 107 ip/ml. Three constructs were tested that contained GFP driven by different promoters: CMV, alpha -elongation factor 1, and neuron-specific enolase. We observed no GFP expression regardless of promoter, titer, or duration of infection in any cell type.

However, with a recombinant, replication-deficient adenovirus that contained GFP driven by a CMV promoter (rAdGFP), we observed robust expression of GFP in both fibroblasts and hair cells but not supporting cells. The following sections elaborate on the infection of hair cells by rAdGFP.

Properties of rAdGFP infection of hair cells

Figure 1A shows a confocal image of a mouse utricle culture viewed from above. The culture was treated with 106 pfu/ml of rAdGFP for 24 h and fixed 36-h postinfection. The sensory epithelium of the utricle contains 600-1,000 hair cells and perhaps an equal number of supporting cells. At the perimeter of the explant are the remnants of the membranous labyrinth that contain fibroblasts. The tissue was stained with rhodamine-conjugated phalloidin to visualize actin, which is particularly dense in the hair bundles and cuticular plates of hair cells. Figure 1B shows GFP fluorescence in the same culture. Within the epithelium hair cells but not supporting cells show GFP fluorescence. On the basis of cell morphology we were able to identify both type I and type II hair cells that were infected. At the periphery fibroblasts expressing GFP were apparent.



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Fig. 1. Confocal images of mouse cochlea and utricle cultures infected with rAdGFP (green) and stained with rhodamine-conjugated phalloidin (red). Each culture was exposed to rAdGFP at the titer noted for 24 h and was then fixed and stained 36 h after the beginning of the infection. A: fluorescence image showing the entire sensory epithelium of a utricle culture stained with rhodamine-conjugated phalloidin to localize hair cells; 737 hair bundles are visible in this image. The scale bar of A also applies to B. B: fluorescence image showing green fluorescent protein (GFP) expression in the same culture as shown in A; 124 hair cells expressed GFP. Hair cells were easily distinguished from fibroblasts that were located only at the periphery of the culture. The culture was exposed to 106 pfu/ml of rAdGFP. C: higher magnification view of a utricle culture exposed to 105 pfu/ml rAdGFP. Few hair cells were infected at this titer. D: utricle culture exposed to 108 pfu/ml rAdGFP; 18 of 27 hair cells in this image expressed GFP. Hair cells in the plane of focus appear the brightest. Those farther from the plane of focus appear more dim. E: fluorescence image of mouse cochlear culture exposed to 106 pfu/ml of rAdGFP counterstained with rhodamine-conjugated phalloidin; 15 of 71 hair cells visible in this image were GFP positive. F: at the periphery of the cochlear culture fibroblasts were also found to express GFP.

The time course of GFP expression was measured by infecting with rAdGFP at a titer of 107 pfu/ml for 24 h, washing with normal medium, and then culturing the utricles for up to 5 days (Fig. 2A). We observed a roughly exponential rise in the number of hair cells expressing GFP that plateaued after ~4 days. The line through the data is an exponential fit with a time constant of 35 h and a maximum of 33% of the hair cells in the epithelium. In other cultures, expression was stable for at least 10 days. Longer times were not tested.



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Fig. 2. Properties of mouse utricle cultures infected with adenovirus. A: time course of GFP expression in cultures exposed to 107 pfu/ml of rAdGFP. Cultures were exposed for 24 h. The number of hair cells expressing GFP was counted at times that ranged from 12 h to 5 days after the onset of rAdGFP exposure. Data are represented as a percentage of 1,000 hair cells in the utricular epithelium. The curve through the data are an exponential fit with a time constant of 35 h. B: percentage of hair cells infected as a function of titer. Epithelia were exposed to rAdGFP at titers that ranged between 104 and 108 pfu/ml for 24 h and maintained in culture for 3 days. The number of hair cells expressing GFP was counted and is represented as a percentage of 1,000 hair cells in the utricular epithelium. The sigmoidal curve through the data yielded a titer of half-maximal expression of 4 × 107 pfu/ml.

To determine the efficacy of adenovirus infection, we tested titers that ranged between 104 and 108 pfu/ml. The cultures were infected for 24 h, and at 3 days postinfection the proportion of hair cells that expressed GFP was measured (Fig. 2B). At 105 pfu/ml a few hair cells expressing GFP were observed (Fig. 1C). At the highest titer we tested, 108 pfu/ml, more than one-half the hair cells were infected (Fig. 1D). The titers we used were not sufficient to infect all hair cells. By assuming that all cells could be infected at higher titers, we fit our data with a sigmoidal curve and calculated a titer for half-maximal expression at 4 × 107 pfu/ml.

Cultures of the mouse cochlea revealed similar expression when exposed to 106 pfu/ml of rAdGFP. In the image shown in Fig. 1E, 15 of 71 (21%) of the hair cells were fluorescent. Although only outer hair cells are fluorescent in this image, inner hair cells were also found to express GFP but at a lower frequency (data not shown). No other cell type was fluorescent except fibroblasts at the periphery of the culture (Fig. 1F).

Viability of infected cells

Although the strong immune response reported for adenoviral vectors used in vivo (Byrnes et al. 1996) presents little concern for our culture system, there was still the possibility that toxic effects could result from the expression of viral genes within the infected hair cells. To address the viability of the cells we used the patch-clamp technique to record both voltage-dependent and transduction currents from rAdGFP-infected hair cells. Individual infected hair cells were identified by GFP fluorescence, and recording electrodes were sealed onto those cells. Figure 3A shows a family of voltage-dependent currents recorded from a fluorescent cell in response to voltage steps that ranged between -124 and +36 mV in 10-mV increments. The large outward currents are similar in both magnitude and kinetics to a delayed rectifier found in normal type II cells of the mouse utricle (Rüsch et al. 1998). The inward currents resembled the fast inward rectifier currents normally found in both type I and type II cells (Rüsch et al. 1998). Normal voltage-dependent currents were recorded from hair cells expressing GFP <= 6 days postinfection (n = 6).



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Fig. 3. Representative whole cell currents recorded from a type II hair cell of a mouse utricule culture exposed to 106 pfu/ml of rAdGFP for 24 h. A: voltage-dependent currents recorded 4 days after exposure to rAdGFP. From a holding potential of -64 mV the membrane potential was stepped from -124 to 46 mV in 10-mV increments. Capacitive transients were removed for clarity. B: transduction current recorded from a GFP-positive hair cell 20 h after exposure to 106 pfu/ml of rAdGFP. Hair bundle deflection was evoked by a fluid jet stimulus. The stimulus was 6 cycles of a 30-Hz, sinusoid with an amplitude of ~2 µm peak-peak (bottom trace). The current and stimulus scale bars and the time axis shown in C also apply to B. C: in the same cell we recorded transduction currents evoked by a 125-ms, ~1-µm step deflection of the hair bundle. Bottom trace: the stimulus waveform.

Perhaps a more rigorous test of the general health of a hair cell is the presence of transduction currents. A fluid-jet stimulus was used to deflect the sensory hair bundles of rAdGFP infected hair cells. We recorded transduction currents evoked by a 30-Hz sinusoidal stimulus (6-cycle burst) from a type II hair cell at 20 h postinfection (Fig. 3B). The currents were asymmetric with an amplitude of ~50 pA peak to peak. Step deflection of the hair bundle evoked a current with a rapid onset that decayed back toward its initial value despite a maintained deflection (Fig. 3C). The current decay, or adaptation, was fit with an exponential function that had a 32-ms time constant. Similar transduction and adaptation were reported previously from uninfected type II hair cells of the mouse utricle (Holt et al. 1997).

However, at times longer than 36 h we observed a detrimental effect of rAdGFP on the hair bundles of infected cells; they were tilted by >45° in the negative direction (toward the shortest stereocilia), a condition never observed in uninfected cells. Figure 4A shows a DIC image from a utricle culture 2 days postinfection. Five healthy-looking hair bundles of noninfected cells surround a single tilted hair bundle of an infected cell. Figure 4, B and C, shows DIC and fluorescence images, respectively, of the same field focused at the cell body layer. It is apparent that the hair cell with the tilted bundle expresses GFP. Almost all infected cells had tilted bundles beyond 36 h postinfection. Although fluid jet-evoked bundle deflections resulted in no transduction currents in the five infected hair cells we tested, the cells were otherwise viable. Normal voltage-dependent currents were recorded as late as 6 days postinfection, and the cell bodies of infected cells appeared healthy for >= 10 days.



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Fig. 4. Effect of rAdGFP on hair bundle integrity. A utricle culture was exposed to 106 pfu/ml for 24 h and imaged 48 h after infection. The scale bar shown in C applies to all images. A: differential interference contrast (DIC) image focused near the base of the hair bundles. Five healthy hair bundles from uninfected cells surround a bundle from an infected cell. Bottom right: another bundle from an infected cell whose cell body does not appear in this set of images. B: DIC image focused at the cell body level. The cell bodies of the 5 uninfected cells and the 1 infected cell (middle) appear healthy. C: fluorescence image showing GFP expression in the infected cell. The GFP fluorescence in this image corresponds to the cell with the tilted hair bundle in A.

Infection with rAd-GFP-Kir2.1

To assess the ability of adenovirus to deliver genes with functional consequences into hair cells we choose to overexpress a K+-selective inward rectifier channel, Kir2.1, for several reasons; Navaratnum et al. (1995) have shown Kir2.1 is expressed in the hair cells of the chick cochlea. The currents predicted to result from overexpression of Kir2.1 should be easily distinguished from the small, endogenous, voltage-dependent currents present at hyperpolarized potentials (Rüsch et al. 1998). Finally, the contributions of inward rectifiers to normal hair cell physiology were well documented for frog (Holt and Eatock 1995), turtle (Goodman and Art 1996), goldfish (Sugihara et al. 1996), and chick (Fuchs et al. 1990; Navaratnum et al. 1995) but are less well understood in mammalian hair cell organs.

Mouse utricle cultures were co-infected with two viruses: 5 × 107 pfu/ml of rAdVgRXR, which contained the gene for the ecdysone receptor under control of a CMV promoter and the gene for the retinoid X receptor under the control of a RSV promoter, and 108 pfu/ml of rAd-GFP-Kir2.1, which contained the Kir2.1 gene fused to the gene for enhanced GFP under control of the inducible ecdysone promoter. Cells infected with rAdVgRXR were expected to express both the ecdysone and retinoid X receptors. To activate the ecdysone receptor complex we added 4 µM of the ecdysone analogue muristerone A after 24. The activated complex consists of a heterodimer that binds the ecdysone inducible promoter of rAd-GFP-Kir2.1. In other systems muristerone A enhances the expression of genes driven by the ecdysone promoter by >30-fold, and protein expression was near half-maximal within 12 h (Johns et al. 1998). Thus cells infected with both rAdVgRXR and rAd-GFP-Kir2.1 and treated with muristerone A were expected to express the GFP-Kir2.1 fusion protein. Because Kir2.1 is a membrane-bound ion channel, we predicted the fusion protein would still be targeted to the cell membrane. Figure 5 shows a ring of fluorescence localized to the cell membrane. Additionally, several punctate regions are visible and may reflect sites of protein synthesis or processing, such as the endoplasmic reticulum or Golgi apparatus.



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Fig. 5. Confocal image of hair from a mouse utricle culture. After a 24-h exposure to 5 × 107 pfu/ml of rAd-RXR, 108 pfu/ml of rAd-Kir2.1-GFP and 4 µM muristerone A induce expression of the Kir2.1-GFP fusion protein. The hair cell is viewed from above focused approximately halfway down the length of the cell body. The protein seems to be localized to the cell membrane and a few punctate regions within the cytoplasm.

To assess the effects of rAd-GFP-Kir2.1 on the electrophysiology of the cells, we first recorded from cells that showed no fluorescence and thus were apparently not infected with rAd-GFP-Kir2.1. Figure 6A shows a family of voltage-dependent currents recorded from a hair cell classified as type I based on the criteria of Rüsch et al. (1998). The instantaneous currents present at the beginning of the voltage steps are evidence of the low-voltage activated K+ conductance (gK,L) present exclusively in type I cells (Rüsch and Eatock 1996a). The data of Fig. 6B are from an uninfected type II hair cell. The outward currents were likely through delayed rectifier K+ channels, and the small inward currents, which were <100 pA at -104 mV, were likely through the endogenous K+-selective inward rectifier channels (Rüsch et al. 1998). Figure 6C shows data from a cell with a visible ring of fluorescence outlining its cell body; on the basis of the shape thus illuminated, it was classified as a type II hair cell. Unlike normal type II cells, voltage steps to -104 mV evoked inward currents of ~2 nA. Taken together the following evidence suggests that these large inward currents were the result of the viral-mediated transfer and expression of the gene for inward rectifier channel, Kir2.1.



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Fig. 6. Voltage-dependent currents recorded from 2 control cells and a cell infected with rAd-Kir2.1-GFP. Currents shown in A-C were evoked by stepping the membrane potential to voltages that ranged between -104 and 46 mV in 10-mV increments. Membrane potentials for select traces are shown to the right. Capacitive transients were removed for clarity. A: currents recorded from an uninfected type I hair cell. B: currents recorded from an uninfected type II hair cell. C: currents recorded from a type II hair cell that based on its fluorescent membrane was infected with rAd-Kir2.1-GFP. Data collected 20 h after introduction of muristerone A to the medium. D: current-voltage relationship for the cells shown in A-C. Steady-state currents were sampled 90 ms after the onset of the voltage steps and are plotted vs. membrane potential. down-triangle, type I cell; triangle , type II cell; , infected type II.

The reversal potential of the peak tail currents after a step to -124 mV was -75 mV (data not shown). The proximity of the reversal potential to EK (-76 mV) indicated the currents were carried primarily by potassium, consistent with the selectivity of Kir2.1 reported by Navaratnum et al. (1995). The average inward rectifier conductance in infected cells was 43 ± 20 nS (n = 6) when fully activated, whereas the inward rectifier conductance from normal type II cells was 3.6 ± 1.7 nS (n = 107) (Rüsch et al. 1998). The outward current at the holding potential (-64 mV) and the large instantaneous currents evoked by voltage steps (Fig. 6C) suggest that a substantial fraction of the Kir2.1 conductance was active at the holding potential. In frog and turtle, inward rectifiers are active at potentials up to ~40 mV positive to the holding potential (Goodman and Art 1996; Holt and Eatock 1995). Thus the input conductance at the holding potential is expected to be larger in a cell that expresses a large inward rectifier conductance. Indeed at -64 mV the infected type II cells had a mean input conductance of 31 ± 6 nS (n = 6), whereas the control type I and type II cells of Fig. 6, A and B, had input conductances of 22 and 0.7 nS, respectively. The physiological consequences of overexpression of Kir2.1 in type II hair cells are consistent with the role of K+-selective inward rectifiers in frog saccular hair cells (Holt and Eatock 1995). These authors showed that hair cells that had the K+-selective inward rectifier conductance, gK1, had zero-current potentials that were more negative than those that did not, -68 versus -50 mV. Figure 6D shows that the zero-current potential (the presumed resting potential) of the infected type II cell was -72 mV (), 17 mV more negative than the uninfected type II cell (-55 mV; triangle ).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adenovirus infects hair cells in vitro

We tested three viral vectors for their ability to infect mammalian hair cells in culture: adenovirus, adeno-associated virus, and HSV. Because our preliminary experiments with rAAV and HSV revealed no expression of reporter genes in hair cells they were not selected for further investigation. However, we cannot rule out the possibility that these vectors may be viable candidates for gene transfer into inner ear hair cells under conditions other than those we tested. On the other hand, we found that adenovirus infected hair cells efficiently and specifically. Approximately one-half of the hair cells were infected at a titer of 4 × 107 pfu/ml; at higher titers most could be infected. Surprisingly, hair cells were much more efficiently infected than other cell types. We observed only scattered fibroblasts that were infected but no other cell type in either cochlear or utricle cultures. In other systems, adenovirus can infect neurons (for review see Chiocca and Cotton 1998). We did not include the nearby vestibular ganglion in these cultures but expect that they would also be infected.

Other viral vectors were also tested for infectivity in the inner ear. In a previous study (Derby et al. 1998) we injected an HSV-1 vector (hrR3) and a vaccinia virus vector (vSC56), both expressing a beta -galactosidase marker, through the round windows of guinea pigs. Both infected a large number of cells in the cochlea, including some in and around the organ of Corti. However, neither infected hair cells at a significant level. Similarly, adeno-associated virus delivered into the scala tympani infected cells in organ of Corti, spiral ganglia, spiral limbus, and spiral ligament but did not infect hair cells (Lalwani et al. 1996). Both studies are consistent with our results in vitro.

Adenovirus was tested in vivo, where it infected a large number of cell types in the spiral ganglia, stria vascularus, spiral ligament, and the lining of the fluid spaces (Raphael et al. 1996; Weiss et al. 1997). In these experiments, it did not seem to infect hair cells. It seems likely that adenovirus finds a receptor for internalization on the apical surface of hair cells, which is a site of substantial vesicle trafficking (Kachar et al. 1997) and which is clearly exposed in culture. Perhaps virus injected into the scala tympani in vivo cannot reach the scala media to gain access to hair cell apical surfaces.

Adenovirus has previously been shown to infect hair cells and other cell types in cultured explants of rat cochlea (Dazert et al. 1997), with infectivity similar to that we find in the mouse cochlea and utricle. In these cultures, which can include the spiral ganglion, neurons were also infected. Dazert et al. (1997) used an adenovirus encoding a beta -galactosidase marker and thus were unable to assess the viability of infected cells while they were still alive.

Cell viability

Cells of the uninfected mouse utricle appear healthy for >= 10 days in culture, and transduction currents can be elicited for at least that long (Holt et al. 1997, 1998). Cells that were infected with adenovirus also appeared generally healthy for 10 days, and currents through voltage-dependent channels appeared normal for >= 6 days. Normal transduction currents were recorded from infected cells during the first 36 h. At longer times, infected cells had altered bundle morphology and lost transduction currents. For many of the studies that would utilize virally mediated gene transfer, 36 h may be enough. Hair cells expressed high levels of GFP within 36 h and would presumably express high levels of a test protein. Thus the effects of the test protein on cell function could be assayed within the first day. On the other hand, some proteins may have detectable physiological effects only after several days; for instance, if proteins are part of a complex then degradation and turnover of existing protein may be required before an effect is observed. For longer-term studies it may therefore be necessary to use a second-generation adenovirus in which most of the adenoviral genes are deleted (Kochanek et al. 1996; Kumar-Singh and Chamberlain 1996). These "gutted" vectors have lower toxicity in other cells and tissues (Schiedner et al. 1998) and probably would in hair cells as well.

Expression of exogenous Kir2.1 in hair cells

As we predicted, adenoviral delivery of the gene for Kir2.1 resulted in the expression of large inwardly rectifying currents in hair cells. Overexpression of Kir2.1 channels altered the normal physiology of the type II cells by shifting the zero-current potential to -72 mV, close to EK (-76 mV). In addition to gK1 contributions to resting potential, Holt and Eatock (1995) showed that a substantial fraction of gK1 was on at rest and that it resulted in a larger input conductance, which in turn resulted in smaller but faster voltage responses to input currents. Thus we expect that the larger input conductance of the type II cells infected with rAd-GFP-Kir2.1 would have a similar effect on their receptor potentials. Indeed Johns et al. (1998) infected cultured superior cervical ganglion neurons with identical viral constructs and showed that the large Kir2.1 conductance decreased the voltage response to injected currents.

To quantify the magnitude of the Kir2.1 protein expression, we compared the mean whole cell conductance (43 nS) recorded from type II hair cells infected with rAd-GFP-Kir2.1 with the Kir2.1 single-channel conductance (17 pS) reported by Navaratnum et al. (1995). By this method, we estimate ~2,500 functional channels were present 20-26 h after introduction of muristerone A; because the channel is tetrameric, we estimate ~10,000 protein monomers. Because this electrophysiological assay of protein expression could only measure functional channels inserted into the membrane, the total amount of GFP-Kir2.1 expressed was presumably greater, perhaps significantly. Thus we can achieve protein expression significant enough to alter hair cell physiology at early times with adenoviral vectors.

The inducible promoter used in this system offers several advantages over a constitutively active promoter. Chief among them is the ability to terminate protein expression by removal of the ecdysone analogue; after allowing time for protein degradation the effects of the induced protein should be reversed (Johns et al. 1998).

Use of gene transfer to elucidate protein function in hair cells

A number of proteins expressed in hair cells were cloned; these include motor proteins (myosin-Ibeta , myosin-VI, myosin-VIIa, myosin XV), ion channels (AChR alpha 9, cSLO, VSCC1D, BIR10, and ROMK1), and transcription factors (Brn 3.1). In addition, some of these are defective in inherited disorders of hearing and balance. Generation of transgenic mice in which these genes are altered would be a traditional approach to study their function, but this method can be time consuming and expensive, and proper interpretation may be confounded by developmental effects. The advantages of adenovirus-mediated gene transfer in vitro, which include rapid construction of viral vectors, ease of viral delivery at high titer, identification of living infected cells, the presence of negative controls in the same tissue, and the ability to disrupt function in fully developed cells, all make this an attractive system with which to study the location, function, and identity of proteins within hair cells.


    ACKNOWLEDGMENTS

We thank X. Breakefield and R. MacDonald for comments on this manuscript and for many helpful discussions. For providing the viral vectors that enabled this study, we thank X. Breakefield (HSV), The University of Florida Vector Core (AAV), and Genezyme (rAdGFP).

This work was supported by National Institute of Deafness and Other Communiations Disorders Grants DC-02281 and DC-00304 to D. P. Corey and DC-02355 to Z.-Y. Chen. D. P. Corey is an Investigator, and J. R. Holt and S. Wang are Associates at the Howard Hughes Medical Institute.


    FOOTNOTES

Address for reprint requests: D. P. Corey, Wellman 414, Dept. of Neurology, Massachusetts General Hospital, Boston, MA 02114.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 31 August 1998; accepted in final form 7 January 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society