Download A modified adenovirus can transfect cochlear hair cells in

Gene Therapy (2001) 8, 789–794
 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00
www.nature.com/gt
RESEARCH ARTICLE
A modified adenovirus can transfect cochlear hair cells
in vivo without compromising cochlear function
AE Luebke1,2, JD Steiger1, BL Hodges3 and A Amalfitano3
1
Department of Otolaryngology and 2Neuroscience Program, University of Miami School of Medicine, Miami, FL; and 3Department
of Pediatrics, Division of Medical Genetics and Department of Genetics, Duke University Medical Center, Durham, NC, USA
The loss of cochlear hair cells, or the loss of their capacity to
transduce acoustic signals, is believed to be the underlying
mechanism in many forms of hearing loss. To develop viral
vectors that allow for the introduction of genes directly into
the cochleae of adult animals, replication-deficient (E1−, E3−)
and replication-defective (E1−, E3−, pol−) adenovirus vectors
were used to transduce the bacterial ␤-galactosidase gene
into the hair cells of the guinea pig cochlea in vivo. Distortion
product otoacoustic emissions, which monitor the functional
status of outer hair cells, were measured throughout the viral
infection periods to identify hair cell ototoxicity. The results
demonstrated that the use of the (E1−, E3−) adenovirus vectors containing CMV-driven LacZ, compromised cochlear
function when gradually introduced into scala tympani via an
osmotic pump. However, when (E1−, E3−, pol−) adenoviral
vectors containing CMV-driven LacZ were used to transduce
cochlear hair cells, there was no loss of cochlear function
over the frequency regions tested, and ␤-galactosidase (␤gal) was detected in over 80% of all hair cells. Development
of a viral vector that infects cochlear hair cells without virusinduced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for
otoprotective strategies to prevent hair cell losses to treat
progressive hearing disorders. Moreover, in vivo (E1−, E3−,
pol−) adenovirus mediated gene-transfer techniques applied
to adult guinea pig cochleae may be useful in testing several
hypotheses concerning what roles specific genes play in
normal cochlear function. Gene Therapy (2001) 8, 789–794.
Keywords: cytomegalovirus promoter; LacZ reporter; replication-defective; cochlea; hair cell; distortion-product otoacoustic
emissions
Introduction
The introduction of genes into cells has become an effective method for the expression of proteins, both for
experimental manipulation and for therapeutic purposes.
One model for manipulating cochlear functions in vivo
relies upon transgenic mouse technologies, that allows
for the over-expression or ‘knock-out’ of gene constructs
potentially critical to cochlea hair cell function. Several
problems with traditional transgenic mouse techniques
include the expense and time involved in isolating the
transgenic lines of mice. In addition, due to the small size
of the mouse cochlea, isolated hair cell experiments are
exceedingly difficult. Finally, the mouse strain most commonly used for the initial recombination events in embryonic stem cells, the 129SvEv strain, is extremely resistant
to noise-damage,1 and thus is not an ideal model system
in which to elucidate all aspects of cochlear function.
Transgenic studies in mice using bacterial artificial
chromosomes (BACs) containing cochlear-specific promoters have directed transgene expression to cochlear
hair cells.2 However, these transgenes are necessarily
expressed throughout development. Thus, it is not clear if
the phenotype exhibited is due specifically to the somatic
Correspondence: AE Luebke, University of Miami Ear Institute (M805),
PO Box 016960, Miami, FL 33101-6960, USA
Received 22 November 2000; accepted 14 February 2001
transgene expression, or if it is a consequence of previously altered cochlear development.
Gene delivery to the nervous system, on the other
hand, offers a potential alternative to traditional transgenic approaches, and has been extensively studied,
primarily using viral vectors. Of the numerous viral vectors tested, only adenoviral vector-mediated gene transfer to cochlear hair cells has been effective in vitro, as herpes simplex virus (HSV) and adeno-associated virus
(AAV) failed to infect cultured hair cells.3–6 HSV, vaccina
virus, lentivirus, and AAV vectors have also failed to
infect cochlear hair cells when tested in vivo.7–9 Interestingly, replication-deficient adenoviral vectors (ie E1−,
E3−), using the Rous sarcoma virus (RSV) promoter to
drive LacZ expression, have failed to transduce cochlear
hair cells in vivo when approximately 107 plaque-forming
units of virus were injected into scala tympani.10 In
addition, the studies using adenoviral constructs to infect
cochlea cells in vivo, either did not assess cochlea function, or used a functional test (ie auditory brainstem
response or ABR) that evaluated principally the ascending auditory system at the auditory nerve and brainstem
level, thus, only grossly examined hair cell function.
In contrast, replication-deficient (E1−, E3−) adenovirus
vectors, when used at higher titers (⬎108 p.f.u. per
cochlea) with the CMV promoter driving green fluorescent protein expression (GFP), have been demonstrated
to effectively transduce cochlear hair cells. Unfortunately,
the latter is achieved at a high price, with a complete loss
Modified adenovirus can transfect cochlear hair cells
AE Luebke et al
790
of outer hair cell function presumably due to the toxic
nature of this class of vector.11 The present study was
designed to determine if a recently described class of
modified adenovirus (E1−, E3−, pol−) vector could be used
to efficiently deliver genes to the cochlea in vivo, without
sacrificing cochlear function. This class of vector is
advantageous in that it has a demonstrated improved
persistence and decreased toxicity in murine models of
hepatic gene transfer.12,13 Since cochlear outer hair cells
are more sensitive than inner hair cells to most of the
common factors that impair hearing (eg excessive
sounds, drug ototoxicity, bacterial or viral diseases, genetic defects, aging, etc), DPOAE testing, which primarily
assesses outer hair cell function, was used to evaluate the
effects of (E1−, E3−, pol−) transduction on peripheral cochlear function. It was anticipated that the outcome of these
experiments would set the course for future interventions
using gene therapy to prevent permanent deafness.
Results
In this current study, the primary aim was to determine
whether the use of an improved adenovirus vector would
be capable of efficiently transducing cochlear hair cells
in vivo, while preserving cochlear function. One of the
principal findings of an earlier study was that an (E1−,
E3−) adenovirus-based vector, when slowly infused into
the scala tympani of the cochlea, was found to be capable
of efficiently transducing cochlear hair cells in vivo, but
also resulted in severely compromised cochlear
function.11
To ensure that there were no deleterious effects on
cochlear function due to the implantation of the perfusion
pump, or the perfusion of substances into the cochlea,
three animals were chronically infused with artificial perilymph for 8 days. These control experiments demonstrated that there was no change in cochlear function as
assessed using DPOAE measures in the form of the DPgram, and shown here for a representative control guinea
pig subject in Figure 1b. Here, the pre- (closed circles)
and post-perfusion (open circles) status of outer hair cell
function, as measured with the DP-gram, are compared.
From these data it can be seen that following perfusion
cochlear function was unchanged, with the 8-day postperfusion DP-gram being similar to the baseline DPgram values.
Figure 2a demonstrates the effect that perfusion of the
replication-deficient (E1−, E3−) adenovirus encoding ␤-gal
had on DPOAE levels for three guinea pigs. When this
virus was perfused into the left ear using titers of 5 × 108
␤-gal-forming units (b.f.u.)/cochlea, the DPOAEs were
reduced, in that by 8 days after virus infection (open
symbols), there were no detectable emissions remaining,
confirming our previous results with the GFP reporter
adenovirus vector.11 In these ears there were no signs of
local inflammatory responses or acute otitis media infections. In contrast, the non-perfused contralateral ears
showed no reductions in DPOAE levels as shown in Figure 2b, suggesting that the vector-induced loss of cochlear function was a locally mediated response directed
only to the perfused ear.
In contrast, when a modified (E1−, E3−, pol−) adenovirus vector encoding LacZ was infused into the left
scala tympani, using titers of 5 × 108 b.f.u./cochlea, of
another three guinea pigs as shown in Figure 2c, there
Gene Therapy
Figure 1 Fluids can be systematically infused into the cochlea without
compromising cochlear function. (a) Schematic showing the placement of
a microcatheter into the basal turn of the left cochlea that connects to an
externalized microperfusion pump. (b) DPOAE measurements in the form
of a DP-gram elicited by equilevel primary tones (ie L1 = L2 = 65 dB SPL)
are shown for an implanted ear of a control experiment guinea pig perfused
with artificial perilymph. These data indicate that there was essentially
no significant changes in DPOAE levels between the 30-min post-pump
(or pre-perfusion) surgical implantation (filled circles) and 8 day postinfusion (open circles) measurements obtained after infusion.
was no significant loss in DPOAEs and hence no change
in cochlear function for up to 8 days (open symbols) following virus infection. As before, the related contralateral
ears also showed no changes in cochlear function
throughout the virus infection period, as seen by the
open-symbol functions of Figure 2d.
To ascertain that the modified (E1−, E3−, pol−) adenovirus actually transduced cochlear hair cells, immunohistochemistry using an antibody directed to ␤-gal was performed on both cochleae from animals that received the
modified adenovirus. These results showed that the
transgene (LacZ) was detected in inner hair cells and
outer hair cells as shown in Figure 3e and f from a crosssection of the cochlear whole-mount shown in Figure 3c.
As the functional integrity of outer hair cells was assessed
using DPOAEs, the focal plane for the whole-mount
photomicrographs of Figure 3a–d are shown at this level.
Outer hair cells within all frequency regions of the cochlear were clearly transduced by the modified adenovirus.
Figure 3a and b display the most apical turn (turn 4) of
the guinea pig cochlea, and Figure 3a provides evidence
that approximately 50% of these apical low-frequency
outer hair cells were transduced by the modified adenovirus, as can be visualized by the specific labeling of the
Modified adenovirus can transfect cochlear hair cells
AE Luebke et al
791
Figure 2 Adenovirus (E1−, E3−) infects the infused cochlea, and compromises cochlear function. (a) Equilevel DPOAE measurements (L1 = L2 =
70 dB SPL) are shown for the left (perfused) and right (non-perfused) ears
of three animals that received (E1−, E3−) adenovirus at 5 × 108
b.f.u./cochlea. (a) Compared with 30 min post-pump implantation (closed
symbols), by 8 days post-infusion (open symbols) there was a significant
loss in DPOAEs in the infused cochlea. (b) In contrast, the non-infused
ears, did not exhibit any significant losses in DPOAE levels suggesting
that the reduced emissions in the perfused ears were due to a local virally
mediated response. (c and d) DPOAE measurements (L1 = L2 = 70 dB
SPL) are shown for the left (perfused) and right (non-perfused) ears of
three animals that received (E1−, E3−, pol−) adenovirus at 5 × 108
b.f.u./cochlea. (c) A modified adenovirus (E1−, E3−, pol−) infected the
infused cochlea without compromising cochlear function. Compared with
30 min post-pump implantation (open symbols), there were no significant
DPOAE changes by 8 days post-infusion (closed symbols). This finding
is in sharp contrast to those with the (E1−, E3−) adenovirus in which the
DPOAEs were reduced and remained at noise floor levels by 8 days postinfusion (panel a). (d) As noted above, there was no significant change in
the DPOAEs measured in the right (non-perfused) ear.
␤-gal protein. Figure 3b shows the equivalent frequency
region in the contralateral cochlea demonstrating no
detectable ␤-gal present in these outer hair cells 8 days
after viral infection.
The mid- to high-frequency regions of the cochleae
showed even greater outer hair cell transduction using
the modified adenovirus as shown by the micrographs of
Figure 3c and d. Approximately 97% of all outer hair cells
in these cochlear regions were transduced by the modi-
a
b
c
d
e
f
Figure 3 Photomicrographs of whole mounts of ␤-galactosidase immunostained (E1−, E3−, pol−) portions of cochlear turns at 8 days post-perfusion
for gp 68 of Figure 2. (a) Adenovirus infection with LacZ expression
driven by the CMV promoter stained outer hair cells (OHCs) in the lowfrequency regions (apical portions) of the cochlea. (b) No LacZ expression
was detected in hair cells of the corresponding right (non-perfused) contralateral ear over the comparable low-frequency regions of the cochlea. (c)
Adenovirus infection with LacZ expression driven by the CMV promoter
outer hair cells (OHCs) in the high-frequency regions (basal portions) of
the cochlea. (d) Again, no LacZ expression was detected in hair cells of the
related right (non-perfused) contralateral ear, over the comparable highfrequency regions (basal portions) of the cochlea. (e and f) Plastic-embedded cross-section of the cochlear whole-mount shown in c. All scale bars
represent 20 ␮m unless noted otherwise.
fied adenovirus. This increase in expression in the basal
cochlear turns is not surprising as the virus was slowly
infused into the scala tympani adjacent to the most basal
turn of the cochlea. Brownian motion and the flow of perilymph from a basal to apical course through the cochlea14 could have contributed to the virus vector diffusion.
Similar to the results shown in Figure 3b with an apical
turn from the contralateral cochlea, there was no transgene expression detected in hair cells of the basal turn of
the corresponding contralateral cochlea as shown in Figure 3d. The percentage of hair cells transduced divided
by the total hair cells present are shown in Table 1 for
both basal (high frequencies) and apical (low frequencies)
cochlear regions.
Discussion
These experiments demonstrated that adenovirus-based
vectors can efficiently transduce cochlear hair cells in
vivo, and that ototoxicity associated with the use of early
generation (E1−,E3−) adenovirus vectors can potentially
be avoided with the use of (E1−, E3−, pol−) adenovirusGene Therapy
Modified adenovirus can transfect cochlear hair cells
AE Luebke et al
792
Table 1 Tabulation of results showing percent (%) transduction, defined as ((No. hair cells transduced/No. total hair cells) × 100) in both
the high (basal) and low frequency (apical) regions of the cochlea, for both adenovirus vectors tested in this study
Adenovirus type
(5 × 108 p.f.u./cochlea)
E1−, E3−, pol−
E1−, E3−
Cochlear
function
+++
−−−
% Transduction basal cochlea
(high frequencies)
% Transduction apical cochlea
(low frequencies)
IHCs (%)
OHCs (%)
IHCs (%)
OHCs (%)
99
13
97
—
90
2
51
0
Both adenovirus vectors were tested at equivalent titers (5 × 108 b.f.u./cochlea) with LacZ as the reporter gene, and the cytomegalovirus
(CMV) promoter driving transgene expression. In all cases, artificial perilymph was used as the carrier solution.
based vectors. The modified adenovirus used here was
previously shown to reduce adenoviral toxicity when
other quantitative measures of virus toxicity (ie liver
enzymes, etc) were measured in mice that had received
(E1−, E3−) and (E1−, E3−, pol−) adenovirus vectors injected
directly into the bloodstream.12
The present study also demonstrated that cochlear
function is a sensitive measure of virus toxicity when
virus is directly infused into scala tympani. Exogenous
DNA can be efficiently delivered and expressed for up
to 8 days in adult cochlear hair cells using (E1−, E3−, pol−)
adenovirus vectors encoding genes important for cochlear function. Longer-term studies are currently
underway to ascertain how long this class of vector persists in guinea pig cochleae. Since many genes have
developmental roles, adenovirus-mediated gene delivery
to adult animals will facilitate experiments on gene function independent of developmental consequences.
Finally, the modified adenovirus perfusion technique
allows expression of a gene specifically in the cochlea,
without expression in other parts of the brain, a problem
that can limit the usefulness of current transgenic technologies.
Previous in vitro experiments using replicationdeficient adenovirus vectors encoding either the CMV
promoter driving the LacZ transgene, or the CMV promoter driving the green fluorescent protein transgene,
also exhibited robust transgene expression when adenovirus vectors were used to transduce cultured hair
cells.3,5 In contrast, previous in vivo studies using adenovirus-mediated gene transfer to hair cells encoding the
RSV promoter driving expression of the LacZ gene failed
to demonstrate efficient transduction of cochlear hair
cells,10,15 which is in contrast to this report. There are a
number of explanations that may account for the divergent results in this study to the previous in vivo adenovirus gene-transfer experiments. Aside from the differences in promoters used, previous adenovirus
transduction attempts utilized titers of adenovirus
(107/cochlea) that were at least 10-fold lower than the
amounts utilized the current study. Furthermore, unpublished studies in our laboratory have confirmed that
attempts of virus infection at lower titers fail to transduce
cochlear hair cells in vivo. Moreover, when cochlear function was monitored using auditory brainstem responses
with low titer adenovirus infection, there was also no loss
of these evoked potentials, which is an indirect indication
that the hair cells were not transduced.16 This outcome
was in contrast to the loss of cochlear function observed
by measuring DPOAEs in the current study using higher
Gene Therapy
titers of (E1−, E3−) adenovirus vectors that were slowly
perfused into the cochlea.
Finally, differences in vector delivery also may have
contributed to the different outcomes between the
present results and those of previous reports. For
example, earlier studies used a 3-min infusion of adenovirus into scala tympani, in contrast to the slow infusion
(1 ␮l/h) of adenovirus into the scala tympani that was
used here. Using this slow-infusion approach, the virus
remained in the catheter for approximately 50 h at 37°C,
yet there was no significant loss of titer (⬍10%) from the
initial titer to the titer after 50 h at 37°C. It has been demonstrated that adenovirus transduction of cells can be
greatly enhanced by increasing contact time of the virus
with the cell,17 which could have also contributed to the
transduction of the sensory hair cells in the current study.
Previous in vivo gene-transfer studies did report transgene expression in spiral ganglion cells and cells of the
cochlear aqueduct in the contralateral ear, an observation
not noted in the present study.18 Possibly the slowinfusion method used here did not approach the pressure
gradients associated with bolus infusions which reduces
the driving force for virus to reach the cochlear aqueduct,
and could account for the observed differences. A benefit
of the perfused ear showing transgene expression with
undetectable expression found in the contralateral ear is
that this approach allows for intra-animal comparisons
for controls. In addition, no transgene expression was
detected in the brains of these animals which further confirms that the technique described here targets virus only
to the perfused cochlea.
In summary, somatic gene transfer and expression of
various genes into the guinea pig cochlear hair cells over
extended time periods can now be envisioned. The welldescribed characteristics of this animal model for studies
of normal and abnormal hearing19,21 will facilitate experimental designs aimed at evaluating both the causes of
hearing loss and the potential to treat human deafness.
Materials and methods
Subjects
The study was performed on 10 adult pigmented guinea
pigs (Ncr/2 strain) weighing 300–350 g and purchased
from the Charles River Laboratories (Boston, MA, USA).
The experimental design consisted of a pre/post-infusion
comparison, with DPOAEs measured before and following perfusion of the adenoviral vectors into the cochlea
using commercially available osmotic pump assemblies
Modified adenovirus can transfect cochlear hair cells
AE Luebke et al
that were chronically implanted as described below.
Additionally, the contralateral cochlea was examined
both functionally, with DPOAEs, and immunohistochemically, for ␤-galactosidase expression (␤-gal), to ensure
that the perfusion was specific to the pump-implanted
cochlea.
Cochlear function assessment using DPOAEs
Before DPOAE testing, the animals were sedated by an
intramuscular (i.m.) injection of 40 mg/kg of ketamine
hydrochloride and 1 mg/kg of acepromazine. DPOAEs at
2f1-f2 were elicited and measured, conventionally, using
equilevel (L1 = L2) primary tones produced by ER-2
speakers and an ER-10B+ microphone assembly (Etymotic
Research, Elkgrove, IL, USA). Stimulus generation and
response acquisition were computer-controlled using an
on-board digital signal processor, along with customized
software controlling stimulus presentation and response
analysis.22 DPOAEs were obtained in the form of
level/frequency functions, or DP-grams (see example in
Figure 1b) for geometric-mean (GM) frequencies (ie (f1 ×
f2)0.5 ) in 0.1-octave steps, from 1.4–17.8 kHz (f2 = 1.8–19
kHz). Primary tone-levels ranged from 45–75 dB SPL, in
systematic 5-dB steps, with f2/f1 = 1.2.
Adenovirus construction and titering
A kanamycin-resistant shuttle plasmid vector was constructed containing the CMV promoter plus the LacZ
gene within the E1 region. The shuttle plasmid was then
digested with PmeI, and electroporated into the BJ5183
recombinogenic strain of E. coli with either the pAdEasy
(E1−, E3−) plasmid, obtained from the Vogelstein Laboratory,23 or the (E1−, E3−, pol−) pAdEDpol plasmid,24 both of
which encode ampicillin resistance genes. The pAdEDpol
plasmid was constructed as follows: the NheI subfragment, encompassing the adenovirus polymerase gene of
the pAdEasy plasmid, was replaced with an identical
subfragment that had been previously modified to
include a 608 bp deletion within the adenovirus polymerase gene. The resultant plasmid was referred to as
pAdEDpol.
After recombination, kanamycin-resistant clones were
screened by BstXI and PmeI digestion to confirm successful generation of the respective full-length recombinant
adenovirus vector genomes. (E1−, E3−) DNA was isolated
and was digested with PacI and transfected into 293 cells.
Similarly, the (E1−, E3−, pol−) DNA was digested with
PacI and transfected into an (E1+, E3+, pol+) expressing
cell line (C-7) that transcomplements the growth of the
polymerase-deleted adenovirus vectors.25 Both adenovirus vectors were then amplified in their respective cell
lines, and confirmed to have the correct construction by
restriction enzyme mapping of the vector genomes. Two
CsCl purifications were performed to generate high titer
vector preparations, followed by a ␤-gal assay using limiting dilutions, to determine the titers of both viruses in
␤-gal forming units (b.f.u.).
Osmotic-pump surgery
The implant surgery, performed under aseptic conditions, secured the osmotic pump (model 2001 (1 ␮l/h),
Alza Co, Palo Alto, CA, USA) in the middle ear. Specifically, the animals were anesthetized using an intramuscular injection of 40 mg/kg ketamine hydrochloride and
5 mg/kg of xylazine. A skin incision was made initially
at the midline to expose the dorsal surface of the skull.
Using a dental burr, a 1.5-mm hole was drilled through
the calvarium at a vertex site 1 cm posterior to the
bregma suture, and a self-tapping stainless-steel screw
was introduced to anchor the pump’s cannula to the
skull. The cannula was fashioned according to the specifications detailed elsewhere,26 and included a drop of silicon at its distal terminus in the cochlear base.
Another incision extending along the dorsal skin to a
post-auricular locus, allowed the exposure of the bullar
portion of the temporal bone. A small defect was created
in the bulla using the tip of a scalpel blade and widened
sufficiently to visualize the round window, as shown in
Figure 1a. The cannula of the pump was filled with the
infusion substance and clamped at the pump end. Using
a fine sharpened metal probe, a small hole was created
through the cochlear bone at its base, and the tip of the
cannula was inserted until the silicon drop was seated
securely against the bone, thus, extending the cannula
about 0.5 mm into scala tympani. A drop of cyanoacrylate cement was applied at the bulla defect to anchor the
cannula at this site. Following confirmation of cannula
placement, the remainder of the defect was covered with
carboxylate cement. To accommodate the body of the
osmotic pump, a subcutaneous pocket was then made
between the scapulae. Before seating the pump, its flow
moderator was inserted into the cannula. To secure the
cannula, its middle portion was looped around the vertex
screw, cemented with methyl methacrylate to the skull
and the skin incision closed.
793
Cochlear perfusions
In all experiments, artificial perilymph (145 mm NaCl, 2.7
mm KCl, 2 mm MgSO4, 1.2 mm CaCl2, 5 mm HEPES
buffer) was used as the carrier solution. Three guinea
pigs received control cochlear perfusions with artificial
perilymph only. The respective adenovirus vectors were
perfused into six guinea pig cochleae using 5 × 108 b.f.u.
of the virus per cochlea. All aspects of this experiment
were reviewed and approved by the University of Miami’s Institutional Animal Care and Use Committee.
␤-Galactosidase immunohistochemistry
After perfusion, the presence of the transgene was
detected using immunohistochemistry for the ␤-gal protein. Immunohistochemistry was used rather than the
enzyme histochemistry, since endogenous ␤-gal activity
present in the guinea pig cochlea (A Luebke, unpublished
observation) prevented detection of ␤-gal activity derived
from the vector genome. In addition, enzyme histochemistry to detect LacZ gene expression has been shown to
underestimate the transfection efficiency.25 Toward this
end, the animals were terminated with an overdose of
pentobarbital, and perfused with 4% paraformaldehyde.
Both cochleae were then harvested and decalcified in 0.1
m EDTA. Following decalcification, the cochlear halfturns were microdissected and processed to detect the
presence of the ␤-gal using an anti-␤-gal antibody
(Promega, Madison, WI, USA). Cochlear half-turns were
incubated overnight in the ␤-gal antibody (1:1000) in 0.01
m PBS at room temperature. After washing with PBS, the
tissues were incubated for 1 h with a donkey-anti-rabbit
secondary antibody (1:800) conjugated to biotin (Jackson
Immunoresearch Laboratories, West Grove, PA, USA),
and then washed and incubated in ABC reagent for 1 h
Gene Therapy
Modified adenovirus can transfect cochlear hair cells
AE Luebke et al
794
(Vector Laboratories, Burlingame, CA, USA), according
to the manufacturer’s specifications. Finally, the tissues
were incubated in diaminobenzidine, cleared in glycerol,
and mounted on depression slides for light-microscopy
viewing. To obtain cross-sections of these whole mounts,
the sections were embedded in plastic (araldite) and cut
into thick sections following the methodology described
by Bohne.28
11
12
13
Acknowledgements
This work was supported by grants from the Public
Health Service DC03086 (AEL), DK52925 (AA), Muscular
Dystrophy Association, USA (AA), and funds from the
University of Miami’s Stanley Glaser Research Foundation and Chandler Chair (AEL). We would like to
thank Dr Ken Muller for his assistance with plastic sectioning.
14
15
16
17
References
1 Yoshida N et al. Acoustic injury in mice: 129/SvEv is exceptionally resistant to noise-induced hearing loss. Hear Res 2000; 141:
97–106.
2 Zuo J, Treadaway J, Buckner TW, Fritzsch B. Visualization of
alpha 9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome. Proc Natl Acad Sci USA 1999; 96: 14100–14105.
3 Dazert S, Battaglia A, Ryan AF. Transfection of neonatal rat
cochlear cells in vitro with an adenovirus vector. Int J Dev Neurosci 1997; 15: 595–600.
4 Staecker H, Gabaizadeh R, Federoff H, Van De Water TR. Brainderived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss. Otolaryngol Head Neck Surg
1998; 119: 7–13.
5 Holt JR et al. Functional expression of exogenous proteins in
mammalian sensory hair cells infected with adenoviral vectors.
J Neurophysiol 1999; 81: 1881–1888.
6 Van de Water TR, Staecker H, Halterman MW, Federoff HJ.
Gene therapy in the inner ear. Mechanisms and clinical implications. Ann NY Acad Sci 1999; 884: 345–360.
7 Derby ML, Sena-Esteves M, Breakefield XO, Corey DP. Gene
transfer into the mammalian inner ear using HSV-1 and vaccinia
virus vectors. Hear Res 1999; 134: 1–8.
8 Hann JJ et al. Transgene expression in the guinea pig cochlea
mediated by a lentivirus-derived gene transfer vector. Hum Gene
Ther 1999; 10: 1867–1873.
9 Luebke AE, Peel AL, Muller CD, Foster PK. DPOAE function
and transgene expression in guinea pig cochlea using AAVdirected gene-transfer methods. Assoc Res Otolaryngol Abstr
1999; 22: 81.
10 Raphael Y, Frisancho JC, Roessler BJ. Adenoviral-mediated gene
Gene Therapy
18
19
20
21
22
23
24
25
26
27
28
transfer into guinea pig cochlear cells in vivo. Acta Otolaryngol
1996; 116: 125–131.
Luebke AE, Steiger JD, Hodges BL, Amalfitano A. Cochlear
function during (E1-) and (E1, E2b-) adenovirus transduction of
guinea pig hair cells. Mol Ther 2000; 1: 580–581.
Hu H, Serra D, Amalfitano A. Persistence of an (E1−,
polymerase−) adenovirus vector despite transduction of a neoantigen into immune-competent mice. Hum Gene Ther 1999; 10:
355–364.
Hodges BL et al. Multiply deleted (E1, polymerase−, and pTP−)
adenovirus vector persists despite deletion of the preterminal
protein. J Gene Med 2000; 2: 250–259.
Salt AN, Inamura N, Thalmann R, Vora AR. Evaluation of procedures to reduce fluid flow in the fistulized guinea-pig cochlea.
Acta Otolaryngol 1991; 111: 899–907.
Yagi M et al. Hair cell protection from aminoglycoside ototoxicity by adenovirus-mediated overexpression of glial cell linederived neurotrophic factor. Hum Gene Ther 1999; 10: 813–823.
Stover T, Yagi M, Raphael Y. Cochlear gene transfer: round window versus cochleostomy inoculation. Hear Res 1999; 136: 124–
130.
Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.
J Virol 1996; 70: 7498–7509.
Stover T, Yagi M, Raphael Y. Transduction of the contralateral
ear after adenovirus-mediated cochlear gene transfer. Gene Therapy 2000; 7: 377–383.
Dallos P. Membrane potential and response changes in mammalian cochlear hair cells during intracellular recording. J Neurosci 1985; 5: 1609–1615.
Preyer S et al. Frequency response of mature guinea-pig outer
hair cells to stereociliary displacement. Hear Res 1994; 77: 116–
124.
Cheatham MA, Dallos P. The dynamic range of inner hair cell
and organ of Corti responses. J Acoust Soc Am 2000; 107:
1508–1520.
Martin GK et al. Locus of generation for the 2f1-f2 vs 2f2-f1 distortion-product otoacoustic emissions in normal-hearing
humans revealed by suppression tuning, onset latencies, and
amplitudes correlations. J Acoust Soc Am 1998; 103: 1957–1971.
He TC et al. A simplified system for generating recombinant
adenoviruses. Proc Natl Acad Sci USA 1998; 95: 2509–2514.
Amalfitano A et al. Production and characterization of improved
adenovirus vectors with the E1, E2b, and E3 genes deleted. J
Virol 1998; 72: 926–933.
Amalfitano A, Chamberlain JS. Isolation and characterization of
packaging cell lines that coexpress the adenovirus E1, DNA
polymerase, and preterminal proteins: implications for gene
therapy. Gene Therapy 1997; 4: 258–263.
Prieskorn DM, Miller JM. Technical report: chronic and acute
intracochlear infusion in rodents. Hear Res 2000; 140: 212–215.
Couffinhal T et al. Histochemical staining following LacZ gene
transfer underestimates transfection efficiency. Hum Gene Ther
1997; 8: 929–934.
Bohne BA. Location of small cochlear lesions by phase contrast
microscopy prior to thin sectioning. Laryngoscope 1972; 82: 1–16.