(Investigative Ophthalmology and Visual Science. 2000;41:580-584.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
The Use of Adenovirus-Mediated Gene Transfer to Develop a Rat Model for Photoreceptor Degeneration
Chooi-May Lai1,
Wei-Yong Shen2,
Ian Constable1 and
Piroska Elizabeth Rakoczy1
1 From the Centre for Ophthalmology and Visual Science, The University of Western Australia;
2 Lions Eye Institute, Perth, Australia.
 |
Abstract
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PURPOSE. To investigate the effects of recombinant adenovirus-mediated
downregulation of cathepsin S (CatS) on the retinal pigment epithelium
and/or neural retina in vivo.
METHODS. The expression of green fluorescent protein (gfp) after subretinal
injection of a recombinant adenovirus, Ad.gfp, into rat eyes was first
established by in vivo fundus fluorescence photography and fluorescence
microscopy. The autofluorescent debris accumulation in Ad.CatSAS
(recombinant adenovirus carrying the antisense CatS gene)-injected rat
eyes was monitored by fluorescence microscopy, and the antisense CatS
RNA expression was demonstrated by in situ hybridization. Changes in
the retinal morphology were assessed by light microscopy.
RESULTS. The gfp expression was present in 30% to 90% of the injection area at
3 days and was absent 9 days after Ad.gfp injection. In
Ad.CatSAS-injected eyes, the expression of antisense CatS RNA was
demonstrated by in situ hybridization. Autofluorescent debris
accumulation was significantly higher in Ad.CatSAS-injected eyes than
in control eyes. The shortening of photoreceptor outer segments in
Ad.CatSAS-injected eyes coincided with intense autofluorescent debris
accumulation. The number of layers of photoreceptor cells decreased
with time and were 11, 9, and 8 at 7, 14, and 28 days after Ad.CatSAS
injection, respectively. In control eyes, the number of layers of
photoreceptor cells (14) remained unchanged.
CONCLUSIONS. These results demonstrate that recombinant adenovirus-mediated
transient modulation of gene expression in retinal pigment epithelial
(RPE) cells could induce changes in the retina, and, in spite of the
low expression of endogenous CatS in RPE cells, this enzyme plays an
important role in maintenance of normal retinal
function.
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Introduction
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Cysteine proteases, cathepsin B, H, L, and S, which are
predominantly present in cells of mononuclear phagocytic origin are
essential for the turnover of intracellular and extracellular proteins.
The inhibition of cysteine protease activity in several organs,
including the eye, has been shown to result in the accumulation of
autofluorescent debris.1
2
The accumulation of
autofluorescent debris in the eye has been linked to several eye
diseases, such as vitelliform macular dystrophy and, possibly, to
age-related macular degeneration. Therefore, the development of models
facilitating studies of autofluorescent debris accumulation in the eye
is of great importance. Traditional peptide-based inhibitors have a
short half-life and, thus, studies of their biologic effect on
lysosomal enzyme inhibition require repeated injections. In this
respect, recombinant adenovirus-mediated gene delivery, which can
ensure transgene expression for an extended period, may offer an
alternative technology for gene expression–related functional studies.
There have been several reports demonstrating efficient transduction of
retinal cells, particularly retinal pigment epithelial (RPE) cells, by
recombinant adenoviruses.3
Recently, our laboratory has
successfully combined recombinant adenovirus-mediated gene delivery
with antisense DNA technol-ogy by constructing a recombinant
adenovirus carrying the cathepsin S (CatS) gene in antisense
orientation (Ad.CatSAS). Ad.CatSAS transduces cultured RPE cells with
high efficiency and downregulates endogenous CatS production in the
transduced cells.4
The present study was conducted to
examine the feasibility of adenovirus-mediated gene delivery for the
development of animal models. The transduction efficiency of this
delivery system is high but transient.3
5
Therefore, in
this work, the longevity, intensity, and area of real-time transgene
expression using Ad.gfp, a recombinant adenovirus construct carrying
the green fluorescent protein (gfp)
gene was first established before investigating if subretinal injection
of Ad.CatSAS could induce any changes in the RPE cell layer or in the
neural retina of the rat eye.
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Materials and Methods
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Construction, Production, and Delivery of Recombinant Adenoviruses
Ad.gfp was constructed by first subcloning the
XbaI/EcoRI fragment of the gfp gene
from the plasmid pEgfp-N1 (Clonetech, Palo Alto, CA) into the
XbaI/EcoRI restricted recombinant adenovirus
vector, pCA13 (Microbix Biosystems, Toronto, Ontario, Canada). The DNA
from the resultant plasmid, pCA13gfp, was then cotransfected with
ClaI-digested Ad5 dl324 DNA into 293
cells, selected, propagated, and purified as described earlier for
Ad.CatSAS and Ad.CatSS.4
The titers used of Ad.gfp,
Ad.CatSAS, and Ad.CatSS were 3 x 1010,
1011, and 1011 plaque-forming units
per milliliter, respectively. Two microliters of each viral preparation
or phosphate-buffered saline containing 10% glycerol (vehicle) was
injected into the subretinal space of 6 week-old normal RCS
rdy+ rats.6
All animal
experimentations adhered to the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research.
Assessment of gfp Gene Expression
The expression of gfp in Ad.gfp-injected eyes (n
= 9) was followed by in vivo fundus fluorescence photography at 3,
9, and 17 days after injection, as previously described.6
The size of the gfp-expressing area within the bleb created
in each eye was assessed by two independent observers. After
confirmation of gfp expression, the rat eyes were either
used for wholemount preparation (n = 6)6
or
processed for plastic-embedding for histologic examination (n
= 3).7
In Situ Hybridization
The sense and antisense digoxigenin (DIG)-labeled CatS riboprobes
were prepared after linearization of the pGem11CatS
plasmid4
with NsiI and HindIII,
respectively. The linearized plasmids were phenol and chloroform
extracted and the sense and antisense probes were transcribed at 37°C
for 2 hours with T7 or SP6 polymerase respectively, in the presence of
DIG-11-uridine-triphosphate using the DIG RNA labeling kit
(Boeh-ringer–Mannheim, Mannheim, Germany). The DIG-labeled
riboprobes were then precipitated, and the efficiency of labeling and
the sensitivity and specificity of the labeled riboprobes were checked
using the protocol provided by the manufacturer. Ten-micrometer-thick
cryosections of eyes enucleated 4 days after subretinal injection of
vehicle or Ad.CatSAS were hybridized with sense and antisense CatS
riboprobes, as previously described.8
The hybridization
temperature was 45°C and the posthybridization wash in 0.1x
SSC/0.1% sodium dodecyl sulfate was performed at 50°C. The
hybridized riboprobes were detected using the Nucleic Acid Detection
Kit (Boeh-ringer–Mannheim) according to the manufacturer’s
protocol.
Evaluation of Retinal Morphology and Fluorescent Debris
Accumulation after Subretinal Injection of Ad.CatSAS or Ad.CatSS
Eyes injected subretinally with Ad.CatSAS (n = 4) and
Ad.CatSS (n = 4) were enucleated 7 days after injection and
were immediately embedded in O.C.T. compound (Sakura Finetechnical,
Tokyo, Japan). Sixty 10-µm thick serial sections of the injection
area were prepared and examined by fluorescence microscopy. The
intensity of the fluorescent signal in sections 1, 30, and 60 was
analyzed by two independent observers and graded from 1 to 3. The
retinal morphology, length of the photoreceptor outer segments (POSs),
and number of layers of photoreceptor cells in Ad.CatSAS- and
Ad.CatSS-injected eyes (n = 3) were assessed by light
microscopic analysis of plastic or paraffin-embedded sections at 7, 14,
and 28 days after injection.7
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Results
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Optimization of Transgene Delivery into the Retina
At 3 days after injection, red-free retinal photography of
Ad.gfp-injected eyes (n = 9) demonstrated no changes in the
retina other than a small bleed around the injection site (data not
shown). The fundus remained normal throughout the course (28 days) of
the study. In vivo fundus fluorescence photography demonstrated a
strong expression of gfp at 3 days after injection (n = 9).
The signal was intense (Fig. 1A
) and covered 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, and 30%,
respectively (mean ± SD of 58.3% ± 22.0%), of the area within
the bleb. By 9 days after injection, the fluorescent signal was
detected in only a few cells, and by 17 days after injection, no signal
was detected (data not shown). Fluorescence microscopic examination of
wholemount preparations of Ad.gfp-injected eyes (n = 6)
showed that most of the gfp-expressing cells were hexagonal RPE cells
(Fig. 1B) . No gfp signal was detected in the neural retina (data not
shown), and not all the RPE cells within the bleb expressed gfp. The
intensity of the gfp signal detected varied from weak (Fig. 1B
, thin
arrows) to strong (Fig. 1B
, thick arrows). Histologic examination of
Ad.gfp-injected eyes (n = 3) showed no signs of morphologic
changes (Fig. 1C)
.
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Figure 1. Characterization of gfp expression in Ad.gfp-injected eyes.
(A) Fundus fluorescence photographic image showing
gfp expression in approximately 60% of the original retinal
bleb at 3 days after injection. Arrow: injection site.
(B) Fluorescence microscopic image showing hexagonal RPE
cells expressing gfp in the wholemounted RPE-choroid-sclera
of the same eye as shown in (A). Thin arrows:
weak gfp signal; thick arrows: strong
gfp signal. (C) Light micrograph of an
Ad.gfp-injected eye with normal retinal morphology at 7 days after
injection. Black arrows: position of RPE layer. Original
magnification: (A) x12; (B) x50; (C) x100.
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Expression of CatS Antisense RNA in the RPE Layer
Hybridization of cryosections of Ad.CatSAS-injected eyes at 4 days
after injection with DIG-labeled sense CatS riboprobe resulted in a
strong signal, seen as a black precipitate, (Fig. 2A , arrows) in the RPE cell layer in and around the injection sites.
RNase treatment of serial sections of Ad.CatSAS-injected eyes before
hybridization with the sense CatS riboprobe resulted in a significant
decrease in the signal intensity (Fig. 2B)
, confirming that the signal
seen in Figure 2A
was of RNA origin. The specificity of the sense CatS
riboprobe was demonstrated by the absence of signal in control
vehicle-injected eyes hybridized with the sense CatS riboprobe (Fig. 2C)
and by the absence of signal in Ad.CatSAS-injected eyes hybridized
with an antisense CatS riboprobe (Fig. 2D)
.
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Figure 2. Light micrographs of cryosections of injected eyes enucleated at 4 days
after injection. (A) Demonstration of antisense CatS RNA
expression in Ad.CatSAS-injected eye hybridized with sense CatS
riboprobe. (B) An RNase-treated section of the eye used in
(A) hybridized with sense CatS riboprobe. Note decrease in
hybridization signal intensity. (C) Section from
vehicle-injected eye hybridized to sense CatS riboprobe. Note absence
of hybridization signal. (D) Hybridization of a section from
an Ad.CatSAS-injected eye with antisense CatS riboprobe. Note absence
of hybridization signal. Arrows: (A)
Hybridization signal in RPE cell layer; (B, C,
and D) RPE cell layer. Magnification, x100.
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The Effect of Ad.CatSAS Delivery on Retinal Morphology
At 7 days after injection, gross morphologic examination of
cryosections demonstrated some changes in the area corresponding to the
bleb generated by the subretinal injection of Ad.CatSAS (Fig. 3A
). In comparison with control vehicle- (data not shown) or
Ad.CatSS-injected eyes (Fig. 3B)
, a significant shortening of
photoreceptor layer was observed in Ad.CatSAS-injected eyes (Fig. 3A
,
double-headed arrow). Fluorescence microscopy of the same sections
before hematoxylin and eosin staining revealed that in
Ad.CatSAS-injected eyes, the morphologic changes closely correlated
with the presence of an autofluorescent signal (Fig. 3C)
. This
fluorescent signal was granular in appearance (Fig. 3C
, white arrows)
and was significantly more intense than the evenly distributed
autofluorescent signal observed in control vehicle-injected (data not
shown) or Ad.CatSS-injected eyes (Fig. 3D
, white arrows and Table 1 ). Both the strong and weak signals observed in Ad.CatSAS- and
Ad.CatSS-injected eyes, respectively, were localized to areas
corresponding to the RPE cell layers marked by the black arrows in
Figures 3A
and 3B
. High-powered microscopic examination of
plastic-embedded and paraffin-embedded sections revealed that the
photoreceptor layers in Ad.CatSAS-injected eyes at 14 days after
injection (Fig. 3E
, double-headed arrow) and at 28 days after injection
(Fig. 3F)
, respectively, were shorter when compared with
vehicle-injected (data not shown) or Ad.CatSS-injected eyes sampled at
14 days (Fig. 3G
, double-headed arrow) and 28 days after injection
(data not shown). The POSs in Ad.CatSAS-injected eyes were not only
shorter (Fig. 3E)
, but they were also disorganized. These changes were
accompanied by a decrease in the number of layers of photoreceptor
cells in the outer nuclear layer (Figs. 3E and 3F)
to 11, 9, and 8
layers by 7, 14, and 28 days after injection, respectively when
compared with the 14 layers present in both vehicle- and
Ad.CatSS-injected eyes throughout the time course. The retina distant
from the injection area, which was not effected by the Ad.CatSAS
injection, remained normal, both in its morphologic and
fluoromicroscopic appearance (data not shown).
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Figure 3. Light micrograph (A, B) and fluorescence
micrograph (C, D) of recombinant adenovirus–injected eyes
at 7 days after injection. (A) Cryosection of an
Ad.CatSAS-injected eye showing photoreceptor loss at 7 days after
injection. (B) Cryosection of an Ad.CatSS-injected eye at 7
days after injection. (A, B) The double-headed
arrows denote the thickness of the photoreceptor layer and
arrows mark position of RPE cell layer. (C)
Fluorescence microscopy of the same cryosection used in (A)
before hematoxylin and eosin staining showing presence of strong,
granular fluorescent material (arrows) in the layer
corresponding to the RPE cell layer (A,
arrows). (D) Fluorescence microscopy of the
cryosection shown in (B) before hematoxylin and eosin
staining detecting a weak and evenly distributed autofluorescent signal
in the layer corresponding to the RPE cell layer (B,
arrows). (E) Plastic-embedded section of an
Ad.CatSAS-injected eye at 14 days after injection. (F)
Paraffin-embedded section of Ad.CatSAS-injected eye at 28 days after
injection. (G). Plastic-embedded section of
Ad.CatSS-injected eye at 14 days after injection. Thickness of the
photoreceptor layer in (E) and (G) is marked by
the double-headed arrow. Arrows: RPE cell
layer. Magnification, (A through D) x50;
(E, F, and G) x100. RD, retinal
detachment; GCL, ganglion cell layer; INL, inner nuclear layer; ONL,
outer nuclear layer.
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Discussion
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The work presented in this study confirmed previous observations
that subretinally injected recombinant adenovirus almost exclusively
transduces RPE cells. This finding suggests that recombinant adenovirus
can be used to target RPE cells without the application of an
RPE-specific promoter. Although adenovirus-mediated transgene
expression is transient, there are a variety of sometimes contradictory
reports in the literature on the longevity of transgene
expression.3
5
To our knowledge, this study, in which fundus fluorescence photography
developed for monitoring adeno-associated-virus–mediated gene
delivery was used,6
9
is the first to report the
noninvasive, real-time monitoring of recombinant adenovirus–mediated
gfp expression in vivo. Our study showed that gfp was expressed at high
enough levels to be detected by in vivo fundus fluorescence photography
from 3 to 9 days after injection. Because our goal was to identify the
longevity of high level of transgene expression, the absence of an in
vivo gfp signal was considered to be the limit of gfp expression. It
was beyond the scope of this study to investigate whether the rapid
decrease in signal intensity was caused by an immune
response10
or by the shutdown of the viral promoter.
Although the expression of gfp was transient, the level of expression
was high, and it was concluded that constant, high-level expression of
biologically active products for up to a week renders this technology
potentially suitable for the development of animal models.
In Ad.CatSAS-injected eyes, the large amount of autofluorescent debris
accumulating in the RPE cells was similar in appearance to the granular
debris induced by cysteine protease inhibitors.1
2
Although the debris induced by cysteine protease inhibitors does not
have the same spectral characteristics as that of
age-related-lipofuscin, it can follow the same route of
compartmentalization in the RPE cells.2
Thus, animal
models based on the forced accumulation of POS-derived debris can be
relevant to the study of the processes occurring in the aging or
diseased human eye.
In a previous study, the accumulation of autofluorescent debris
in animals injected with cysteine protease inhibitors was reported to
be accompanied by some disorganization in the POS layer.2
Compared with these protease inhibitor studies, the neural retinal
changes observed in Ad.CatSAS-injected animals were more pronounced.
These changes included the shortening of the POSs and a decrease in the
number of layers of photoreceptor cells in the outer nuclear layer,
which were not observed in vehicle-, Ad.gfp-, or Ad.CatSS-injected
animals. Therefore, it can be concluded that these changes were induced
by Ad.CatSAS. Protease inhibitor uptake is not a cell-specific process,
thus protein inhibition studies were unable to determine whether
changes in the photoreceptor layer were due to inhibition of neural
retinal cysteine protease activity or to the breakdown of RPE function.
Because subretinally delivered recombinant adenoviruses specifically
transduce RPE cells,3
and the effect of the antisense RNA
is limited to the transduced cells, it is thus suggested that the
neural retinal changes observed are secondary effects of the
downregulation of CatS activity in the RPE cell layer. From this study,
a correlation between Ad.CatSAS delivery and photoreceptor degeneration
was established. We propose that further studies be performed to
elucidate the exact role of CatS, either by the use of a recombinant
adeno-associated virus that can mediate long-term downregulation of
CatS activity or by the generation of knockout mice.
The transient downregulation of CatS activity in RPE cells in a
rat model has been shown to induce the accumulation of autofluorescent
debris and to compromise retinal morphology. This implies that CatS may
play an important role in the maintenance of normal retinal function.
Furthermore, these results demonstrate that recombinant
adenovirus-mediated transient gene expression in RPE cells can be used
to induce changes in the retina and therefore to provide an
understanding of the role of certain genes in the maintenance of normal
retinal function.
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Acknowledgements
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The authors thank Louise Kemp, Katrina Spilsbury, and Meaghan Yu
for help in preparing the manuscript, and Meditech Research Ltd. for
their support.
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Footnotes
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Supported by the National Health and Medical Research Association of Australia.
Submitted for publication December 29 1999; revised May 6 and July 9, 1999; accepted July 14, 1999.
Commercial relationships policy: N.
Corresponding author: Piroska Elizabeth Rakoczy, Centre for Ophthalmology and Visual Science, 2 Verdun Street, Nedlands, Perth, Western Australia. rakoczy{at}cyllene.uwa.edu.au
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References
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Katz, ML, Shanker, MJ (1989) Development of lipofuscin-like fluorescence in the retinal pigment epithelium in response to protease inhibitor treatment Mech Ageing Dev 49,23-40[Medline][Order article via Infotrieve]
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Ivy, GO, Kanai, S, Ohta, M, et al (1989) Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine protease inhibition Adv Exp Med Biol 266,31-44[Medline][Order article via Infotrieve]
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Bennett, J, Wilson, J, Sun, D, Forbes, B, Maguire, A. (1994) Adenovirus vector-mediated in vivo gene transfer into adult murine retina Invest Ophthalmol Vis Sci 35,2535-2542[Abstract/Free Full Text]
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Rakoczy, PE, Lai, M, Baines, M, Spilsbury, K, Constable, I. (1998) Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells Invest Ophthalmol Vis Sci 39,2095-2104[Abstract/Free Full Text]
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Kumar–Singh, R, Farber, DB (1997) Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration Hum Mol Genet 38,2857-2863
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Rolling, F, Shen, W, Tabarias, H, et al (1999) Evaluation of AAV mediated gene transfer into the rat retina by clinical fluorescent photography Hum Gene Ther 10,641-648[Medline][Order article via Infotrieve]
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Shen, W, Yu, M, Barry, C, Constable, IJ, Rakoczy, PE (1998) Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat Br J Ophthalmol 82,1063-1071[Abstract/Free Full Text]
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Garrett, KL, Anderson, J. (1995) Colocalization of bFGF and the myogenic regulatory gene expression in dystrophic mdx muscle precursors and young myotubes in vivo Dev Biol 169,596-608[Medline][Order article via Infotrieve]
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Bennett, J, Duan, D, Engelhardt, JF, Maguire, A. (1997) Real-time noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction Invest Ophthalmol Vis Sci 38,2857-2863[Abstract/Free Full Text]
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Hoffman, LM, Maguire, AM, Bennett, J. (1997) Cell-mediated immune response and stability of intraocular transgene expression after adenovirus-mediated delivery Invest Ophthalmol Vis Sci 38,2224-2233[Abstract/Free Full Text]
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Abstract
Introduction
