Download Neuroscience Letters Efficient and specific transduction of cochlear

Neuroscience Letters 442 (2008) 134–139
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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Efficient and specific transduction of cochlear supporting cells by
adeno-associated virus serotype 5
Ester Ballana a,1 , Jing Wang b , Frédéric Venail b , Xavier Estivill a,c , Jean-Luc Puel b ,
Maria L. Arbonès a,d , Assumpció Bosch e,∗
a
Genes and Disease Programme, Center for Genomic Regulation (CRG), UPF, Barcelona, Catalonia, Spain
INSERM U583, Montpellier, France
Centre for Biomedical Research on Epidemiology and Public Health (CIBERESP), Barcelona, Catalonia, Spain
d
Centre for Biomedical Research on Rare Diseases (CIBERER), Barcelona, Catalonia, Spain
e
Department of Biochemistry and Molecular Biology, Center for Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona,
E-08193 Bellaterra, Catalonia, Spain
b
c
a r t i c l e
i n f o
Article history:
Received 10 April 2008
Received in revised form 19 June 2008
Accepted 19 June 2008
Keywords:
Gene transfer
AAV5 serotype
Inner ear
Supporting cells
a b s t r a c t
Congenital deafness, affecting 1 in 1000 neonates, can lead to major problems in speech, cognitive and psychosocial development. Congenital deafness is mainly caused by mutations in connexins, hemi-channel
proteins forming gap-junctions between supporting cells in the sensory epithelia. We describe a high
tropism of AAV5 serotype for the supporting cells of the cochlea, both in vitro in postnatal day 4 mouse
explants, and in vivo in the adult guinea-pig inner ear, through scala media perfusion. AAV5 transduction correlates with PDGFR␣ expression, previously reported as AAV5 receptor. This vector could be of
major interest in addressing gene therapy approaches to deafness as well as for studying basic aspects of
inner-ear development and hearing mechanisms.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Hearing impairment is a clinical alteration affecting more than
250 million people worldwide. Among these, one child in a thousand before the age of two can suffer from congenital deafness,
which may lead to significant problems in speech, cognitive and
psychosocial development [21]. Sixty percent of these cases have
a genetic cause and no efficient treatment has yet been found.
Most of the pathologies leading to hearing impairment affect
the inner ear, where only 15,000 neurosensorial cells – the hair
cells – establish connections with 30,000 primary hearing neurons. However, the main cause of congenital deafness is due
to mutations in connexins (CX), mainly expressed in the supporting cells of the cochlea [10,14]. Connexins are hemi-channel
proteins forming gap-junctions between supporting cells in the
sensory epithelia. In many populations, mutations in CX26 [14],
CX30 [11] and in other CX genes [24] account for about half of
the inherited prelingual non-syndromic cases of deafness. Despite
recent surgical and technological advances, there is still no clinically efficient treatment for hearing disorders. Gene therapy could
∗ Corresponding author. Tel.: +34 935814203; fax: +34 935814200.
E-mail address: [email protected] (A. Bosch).
1
Present address: IrsiCaixa Foundation, Hospital Universitari Germans Trias i
Pujol, Badalona, Catalonia, Spain.
0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2008.06.060
potentially offer a beneficial approach to solving this type of deafness.
The inner ear offers several advantages as a model for gene therapy, as it is a small and well-compartmentalized receptacle that
is easily accessible through retroauricular injection; additionally,
cochlear endolymph and perilymph volumes are well characterized in humans and in animal models, so adverse effects of high
volume and pressure can be avoided.
Several publications have reported viral and non-viral transduction of the inner ear in rodents through different routes
of administration, targeting a range of cell types with variable
efficiencies [3,7,15,16,20,25]. Among the viral vectors available,
adeno-associated virus (AAV) is one of the most promising gene
therapy vectors for human clinical trials. Different serotypes of
AAV vectors have shown high transduction specificity for certain
cell types. For example, AAV1 and AAV5 mostly infect neurons in
the central nervous system, and are able to diffuse into the brain
parenchyma for several millimetres, while AAV4 infects ependymal
cells [4,6].
In order to ascertain an AAV serotype with a high and specific
tropism for supporting cells in the cochlea, we tested the infection
capacity of different pseudotyped AAV vectors in mouse organotypic cochlear cultures, and in vivo in the adult guinea-pig inner
ear and we characterized the cell types transduced in both models.
E. Ballana et al. / Neuroscience Letters 442 (2008) 134–139
All animal procedures involved were previously approved by
the Animal Ethics Committees of Catalonia and France and were
performed in accordance with recommendations for the proper
care and use of laboratory animals. Cochleae were dissected from
postnatal day 3 CD-1 mice. Epithelia of the entire cochleae were
infected in eppendorf tubes containing 20 ␮l of culture medium
and virus particles of AAV1, AAV2, AAV4 or AAV5 at a final concentration of 109 viral genomes/ml (vg/ml) for 12 h, at 37 ◦ C and 5%
CO2 . Subsequently, explants were cultured on laminin-coated LabTek 8-well culture slides (Nalge Nunc) for 5, 10 or 15 days in DMEM
supplemented with d-glucose (8.25 mM), l-glutamine (1 mM) and
N1 (1:100) (Sigma).
For cochlear explant immunostaining, we used antibodies
against glial fibrillary acidic protein (GFAP) (1:400; Dako), Cx26
(1:200; Zymed), Neurofilament 200 kDa (NF200) (1:200, Sigma),
PDGFR␣ (1:200; Santa Cruz Biotechnology), myelin basic protein (MBP) and peripherin (1:200; Chemicon). Primary antibodies
were detected with secondary antibodies conjugated with Alexa
Fluor 488 or 633 (1:1000; Molecular Probes) and cultures were
counterstained for F-actin with rhodamine-conjugated phalloidin
(100 mM; Sigma). Whole mounts were observed using fluorescence
and/or confocal microscopy (Leica).
Immunohistochemistry on adult guinea-pig cochleae was performed for PDGFR␣ on 10 ␮m-thick cryo-sections. Double labeling
was performed using antibodies against calbindin (1:100, Chemicon), myosin VIIa (1:200, ABR), and NF200 (1:800). Secondary
labeling with Alexa Fluor 594 and 647 (1:500, Molecular Probes)
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was used to localize these proteins with a Bio-Rad confocal microscope.
For in vivo cochlear gene transfer, adult guinea-pigs were anaesthetized and endolymphatic perfusion was performed by shaving a
small fenestrate in the cochlear bony capsule of the basal turn over
the stria vascularis. A second aperture was made at the cochlear
apex. Endocochlear potentials were recorded to ensure the correct
positioning of the aperture. Only those animals with normal endocochlear potentials following this procedure (95.4 mV ± 1.1, n = 5)
received AAV5 vectors.
Vectors at titers of 3.7 × 109 vg diluted in 100 ␮l of artificial
endolymph were perfused through the scala media fenestration
in the basal turn, and flowed out of the cochlea through the opening at the apex. Artificial endolymph composition was NaCl 1 mM,
KCl 126 mM, KHCO3 25 mM, MgCl2 0.025 mM, CaCl2 0.025 mM,
K2 HPO4 1.4 mM (pH 7.4; osmolarity = 300 mOsm/kg H2 O). Animals
were euthanized 1 month after perfusion.
AAV1, AAV2, AAV4 and AAV5 pseudotypes containing the
CMVeGFP cassette flanked by AAV2-ITR sequences and the capside for each serotype were used to infect P4 mouse inner-ear
explants at titers of 109 vg/ml. After 15 days in culture, cochleae
counter-stained with phalloidin showed different patterns of transduction, depending on the serotype (Fig. 1). Fibroblast-like cells
were infected with AAV2 (not shown) and AAV4 (Fig. 1C, arrows).
AAV1 and, more efficiently, AAV2 were able to infect hair cells
(Fig. 1A and B, arrowheads). In addition, and in accordance with that
reported by other authors [16,20], AAV1 also infected supporting
Fig. 1. Differential pattern of AAV pseudotyped-mediated transduction of mouse cochlea. Mouse P4 explants infected with AAV2/1 (A), AAV2/2 (B), AAV2/4 (C), or AAV2/5
(D) containing a CMVeGFP cassette (green) and counterstained with rhodamine-conjugated phalloidin (red) after 15 days in culture. Arrowheads indicate infected hair cells,
arrows indicate fibroblast-like cells and asterisks indicate position of outer and inner hair cell rows at low magnification. OHC, outer hair cells; IHC, inner hair cells; HC, hair
cells. Scale bar: 25 ␮m.
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E. Ballana et al. / Neuroscience Letters 442 (2008) 134–139
cells in the cochlea (Fig. 1A). In contrast, AAV5 showed hightransduction specificity for a particular structure located between
the hair and the ganglion cells (Fig. 1D). Additional experiments
performed with half-log lower titer AAV5 showed the same pattern of transduction at a similar efficiency (data not shown). With
shorter virus incubation times (10 days), we obtained the exact
transduction pattern. At 5 days, however, the level of GFP expression was too low to identify this structure (data not shown). Up to
40 cochleae were infected, with similar results.
To identify the AAV5-transduced cells, we performed immunohistochemistry with antibodies targeting different types of
inner-ear cell. We did not observe expression of GFP in immunoreactive cells for anti-peripherin (Fig. 2A) or anti-NF200 (Fig. 2B),
which label type II and type I neurons, respectively; nor for
anti-myosin VIIa (data not shown), which labels hair cells. GFPexpressing cells in the cochlea were always detected in the same
structure in which GFAP-expressing supporting cells are located
(Fig. 2C and D). In the mouse cochlea, all supporting cells express
GFAP at P3 in a gradient decreasing in intensity from base to apex
[19]. Interestingly, AAV5-transduced cells are also more numerous
in the base than in the apex of the cochlea. To identify the type of
supporting cells expressing GFP, we used anti-MBP and anti-Cx26
antibodies. These antibodies labeled most of the supporting cells
interdigitated with hair cells in the sensory epithelia of the cochleae
at time of infection (P3–P4) (Fig. 3A and B). Unfortunately, neither of
these two antibodies worked in explants that had been in culture for
several days, when GFP was evident. As many other viruses, AAV5
requires charged carbohydrates, in particular ␣-2,3-N-linked sialic
acid, for efficient binding and transduction [13]. However, the only
membrane receptor reported so far for AAV5 is PDGFR␣, described
in the neurons of the rat hippocampus [8] and in the rods of the
human retina [17]. Immunohistochemistry for PDGFR␣ performed
in P4 mouse cochleae showed immunoreactivity in MBP-positive
and Cx26-positive cells, consistent with the expression of the AAV5
receptor in most of the cochlear supporting cells (Fig. 3C). Our
results therefore provide evidence that AAV5 is able to specifically infect the supporting cells of the cochlea in postnatal mice,
probably through the PDGFR␣. Receptor expression can be speciesdependent, and can be modified throughout cochlear development;
or it may be altered when a tissue is cultured for several days.
To address these possibilities, we characterized the expression of
PDGFR␣ in the adult guinea-pig, an animal model extensively used
for inner-ear studies. We found PDGFR␣ immunoreactivity in different types of supporting cells, including Deiter’s, Hensen’s, pillar
and the phalangeal cells of the guinea-pig organ of Corti (Fig. 4A).
This result correlated with the in vitro studies carried out on mouse
P4 inner ear. In addition, PDGFR␣ immunoreactivity was detected
in spiral ganglion neurons and in the marginal cells of the stria
vascularis in the adult guinea-pig (Fig. 4A and B).
Expression of viral receptors is needed for vector-cell transduction; we therefore hypothesize that if we could access the
supporting cells in the guinea-pig cochlea we would be able
to transduce them with AAV5. Scala media perfusion in adult
guinea-pigs of 3.7 × 109 vg/cochlea showed GFP expression in the
supporting cells of the cochlea, mainly in Deiter’s, pillar and inner
border cells, correlating with PDGFR␣ immunoreactivity (Fig. 4C).
Fig. 2. AAV2/5 specifically transduces supporting cells of the mouse cochlea. Confocal images of cochlear explants processed for immunofluorescence at 10 days post-infection
with AAV2/5-CMVGFP and labeled with anti-peripherin, labeling type II neurons (A), anti-NF200, labeling type I neurons (B) (both in blue) or anti-GFAP, labeling supporting
cells (red) (C and D). A higher magnification of (C) is shown in (D). (A and B) were counterstained for F-actin with rhodamine-conjugated phalloidin (red). HC, hair cells. Scale
bar: 25 ␮m.
E. Ballana et al. / Neuroscience Letters 442 (2008) 134–139
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Fig. 3. Supporting cells in the neonatal mouse cochlea expressed PDGFR␣. Freshly isolated cochlea from P4 mice were processed for immunofluorescence using anti-MBP
(A), anti-Cx26 (B) and anti-PDGFR␣ (C). All tissues were counterstained with rhodamine-conjugated phalloidin (red). Arrowheads indicate supporting cells. OHC, outer hair
cells; IHC, inner hair cells. Scale bar: 25 ␮m.
However, no neurons were transduced, probably due to the difficulty of reaching the spiral ganglion neurons through scala media
perfusion. In addition, some GFP expression was seen in marginal
cells of the stria vascularis that also expressed PDGFR␣ (Fig. 4D).
Sporadically, we were also able to target PDGFR␣-negative inner
hair cells which may express an alternative, still unknown, AAV5
receptor (Fig. 4C).
Other authors have reported AAV5 transduction in rodent
cochleae, both in organotypic cultures from postnatal animals and
in vivo, in adult mice, with different results [9,16,20]. While Di
Pasquale et al. found only 1% transduction of vestibular hair cells
in P2 rat explants using a CMV promoter [9], Stone et al. reported
very rare transduced-hair cells using a chicken ␤-actin promoter
and rare transduced-interdental and Hensen cells with a GFAP promoter in mouse P1 organotypic cultures [20]. In vivo round window
transduction of adult mouse cochleae using another ubiquitous
promoter, chicken ␤-actin, mostly showed infection of inner hair
and spiral ganglion cells, and at lower levels, of inner sulcus, spiral limbus, and Claudius’ cells [16]. In these reports virus load
was at least 10 times higher than in the present work with lower
transduction efficiencies in supporting cells, suggesting that the
increased tropism we report here for this particular cell type is not
due to saturation of viral load. Moreover, we showed correlation
between PDGFR␣ and AAV5 transduction, indicating specificity of
AAV5 infection through its receptor in the supporting cells of the
cochlea. Length of expression could also account for some of the
differences reported. Other authors analyzed rodent explants at
day 5 or at day 8 post-infection [9,20]; in contrast, we waited until
day 15, described as the maximum for GFP expression in organotypic cultures of human airway epithelia [26]. We did not observe
fluorescence in explants 5 days post-infection, indicating that supporting cells may need longer exposures to show AAV5-driven GFP
expression. Similarly, we evaluated the in vivo experiments 4 weeks
after transduction, whereas Liu et al. analyzed AAV5 expression at
day 10 and recorded few transduced supporting cells [16]. Most of
the in vivo experiments performed with AAV5 were evaluated at
least 3 weeks after infection in a wide variety of tissues, including the CNS [4,6]. In the retina, AAV5 is one of the most efficient
AAV serotypes and its expression starts at 2 weeks, although it
peaks between 3 and 4 weeks [2,18]. Thus, it is possible that tissues processed at 10 days did not show enough reporter protein to
highlight transduced supporting cells in the cochlea. Nevertheless,
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E. Ballana et al. / Neuroscience Letters 442 (2008) 134–139
Fig. 4. Localization of PDGFR␣ in adult guinea-pig cochlea and transgene distribution after in vivo delivery of AAV2/5 in the inner ear target supporting cells. Immunohistochemistry for PDGFR␣ on adult guinea-pig cochlea. (A and B) Confocal images of transverse (midmodiolar) section through the medial turn in the adult guinea-pig cochlea,
showing immunoreactivity to PDGFR␣ (red). In (A), calbindin counterstains hair cells in blue and NF200 counterstains nerve fibers of spiral ganglion neurons in green. PDGFR␣
was localized in the Deiter’s cells (DCs), Hensen’s cells (HCs), inner pillar cell (ip), outer pillar cell (op) and phalangeal and border cells (asterisks). PDGFR␣ immunoreactivity
was also present in the marginal cells of the stria vascularis (sv) (B). (C and D) Confocal microscopy of sections of an AAV5-perfused adult guinea-pig cochlea at 1 month. GFP
expression (green) was colocalized with myosin VIIa (red) in the inner hair cell (IHC) (C). GFP expression was also observed in the supporting cells of the organ of Corti such
as Deiter’s cells (DCs) (C). GFP expression also occurred in the marginal cells of the stria vascularis (sv) (D). IHC, inner hair cell; OHCs, outer hair cells; sl, spiral ligament; nf,
nerve fibers; isb, inner spiral bundles; tC, tunnel of Corti. Scale bar: 10 ␮m.
high transduction efficiencies in inner hair cells and spiral ganglion
neurons were reported [16]. We also observed transduction in inner
hair cells, but not in neurons, although they were positive for the
AAV5 receptor. These differences may also be due to the delivery
route or the administration volume.
Mice models deficient in Cx26 or Cx30 show hearing impairment [5,22] and they are of interest for assaying gene therapy
approaches with AAV5 coding for connexin genes. In addition, overexpression of Cx26 has been shown to correct Cx30-linked deafness
by crossing two genetically modified mice models, suggesting that
up-regulation of Cx26 could be used as treatment for deafness
caused by Cx30 mutations [1]. However, it is not known when Cx26
needs to be overexpressed in order to correct Cx30 deficiency. A
question that requires further attention is whether connexins may
already be needed early during development or in the postnatal
period. This issue might be addressed by specific administration of
an AAV5 coding for Cx26 to the supporting cells of Cx26 or Cx30deficient mouse models at different time points.
Unlike avian hair cells, mammalian hair cells do not regenerate
and the initial population of 15,000 cells per cochlea is continuously diminished through frequent damage such as loud sounds,
certain drugs, infections or the natural aging process. Recently,
mammalian cochlear supporting cells have been shown to be able
to divide and trans-differentiate into hair cells when expressing
genes involved in hair-cell differentiation such as Math1/Atoh1
[12], or when cyclin-dependent kinase inhibitor p27Kip1 is downregulated [23]. These results suggest that postnatal mammalian
supporting cells are potential targets for therapeutic manipulation.
Specifically targeting supporting cells in the cochlea with AAV5
coding for Math1 or containing RNA interference sequences against
p27Kip1 may therefore restore some of the lost hair cells in the
cochlea.
In conclusion, we demonstrate specific AAV5 tropism for the
supporting cells of the cochlea both in vitro, in mouse explants, and
in vivo, in the adult guinea-pig. This vector could be of considerable
interest in addressing gene therapy approaches to deafness, but also
in the study of basic aspects of inner-ear development and hearing
mechanisms.
Acknowledgements
We thank the vector core of the University Hospital of Nantes
that was supported by the Association Française contre les
Myopathies (AFM) for providing the AAV vectors. EB was a recipient
of a fellowship from the Generalitat de Catalunya (2003FI00066)
and AB was an investigator of the Reincorporación de Doctores y Tecnólogos and the Ramon y Cajal Programs from the Spanish Ministry
of Education and Science. This work was supported by the Gen-
E. Ballana et al. / Neuroscience Letters 442 (2008) 134–139
eralitat de Catalunya (ACI2001/8), the Instituto de Salud Carlos III
(G03/203, PI05-2347, PI05-1705) and the Fundació Marató de TV3
(98/1710).
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