1Department of Neurobiology,
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
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 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.
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) 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 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),
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 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 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 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 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 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.
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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-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.
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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). 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.
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
. 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.
), 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.
-elongation factor promoter at 1011 ip/ml
(Zolotukhin et al. 1996
), and neuron-specific promoter at 1010 ip/ml (Peel 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.
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.
, 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 M
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.
; 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
107 ip/ml. Three constructs were tested that contained
GFP driven by different promoters: CMV,
-elongation factor 1, and
neuron-specific enolase. We observed no GFP expression regardless of
promoter, titer, or duration of infection in any cell type.
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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.
|
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).
|
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.
|
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.
|
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.
|
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;
).
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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
-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
-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-I, myosin-VI, myosin-VIIa, myosin XV), ion channels (AChR
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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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