 |
|
Combined Effect of Cyclosporine and Sirolimus on Improving the Longevity of Recombinant Adenovirus–Mediated Transgene Expression in the Retina
Wei-Yong Shen, MD;
May C. Lai, PhD;
John Beilby, MD;
Nigel L. Barnett, PhD;
Jie Liu, MD;
Ian J. Constable, MD;
Piroska E. Rakoczy, PhD
Arch Ophthalmol. 2001;119:1033-1043.
ABSTRACT
| |
Objectives To reevaluate the longevity and intraocular safety of recombinant adenovirus
(rAd)–mediated gene delivery after subretinal injection, and to prolong
transgene expression through the combination of 2 synergistic immunosuppressants.
Methods An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression
was monitored in real time by fundus fluorescent photography. Intraocular
safety was examined by observation of changes of retinal pigmentation, cell
infiltration in virus-contacted area, immunophenotyping for CD4+
and CD8+ cytotoxic T lymphocytes, and CD68+ macrophages,
histologic findings, and dark-adapted electroretinography. Two synergistic
immunosuppressants, cyclosporine and sirolimus, were used alone or in combination
to prolong transgene expression by temporary immunosuppression.
Results The GFP expression peaked on day 4, dramatically decreased on day 10,
and was not detectable on day 14. The decreased GFP expression was coincident
with cell infiltration in virus-contacted area. Immunostaining showed that
the infiltrating cells were CD4+ and CD8+ cytotoxic
T lymphocytes and CD68+ macrophages. Clumped retinal pigmentation
and decreased b wave of dark-adapted electroretinogram were observed at 3
to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal
degeneration. Transient immunosuppression by cyclosporine and sirolimus, either
alone or in combination, improved transgene expression, with the combination
being the most efficient. The combined immunosuppression attenuated but did
not retard the rAd-induced retinal damage.
Conclusions Transgene expression mediated by rAd after subretinal delivery is short-term
and toxic to the retina. Combination of cyclosporine and sirolimus may act
as an immunosuppressive adjunct to prolong rAd-mediated gene transfer.
Clinical Relevance The intraocular safety of rAd should be carefully considered before
clinical trials are performed.
INTRODUCTION
GENE THERAPY, which has the ability to produce high concentrations of
therapeutic agents in site for sustained periods, has the potential to change
traditional treatments for human diseases. The eye is an ideal target for
gene therapy because it is separated from other organs by the blood-retinal
barrier, there are a limited number of cells confined to a small space, and
transgene expression can be monitored optically without invasive interventions.1-3
Recombinant adenovirus (rAd) is one of the most widely used viral vectors
targeting both resting and proliferating cell types. In the eye, subretinal
delivery of rAd almost exclusively transduces the retinal pigment epithelium
(RPE) with high efficacy.2, 4-5
The preferential transduction of RPE cells is potentially useful to develop
novel strategies for the treatment of retinal degenerations.6-7
It also seems realistic that delivery of neurotrophic factor genes to the
RPE cells could attenuate the progress of retinitis pigmentosa.8-9
However, the longevity of rAd-mediated transgene expression in the eye dramatically
varied from days to months in previous studies.2, 4-5,7
The use of green fluorescent protein (GFP) as a reporter gene may allow us
to noninvasively clarify this issue in living animals.
Host immune responses to E1 and E3 region deleted rAds currently present
a general problem for long-term gene delivery. Cytotoxic T lymphocytes have
been shown to mediate the destruction of rAd-transduced cells. Humoral response
leads to the production of antibodies, inhibiting further delivery of rAd.10-11 Traditionally, the eye has been considered
an immune-privileged site. However, the intraocular safety after subretinal
delivery of rAd is controversial.12-14
Recent data indicate that both humoral and cellular immune responses are evoked
by rAd directly delivered into the subretinal space.14-15
This raises the possibility that suppression of the host immune system may
significantly improve the efficacy of transgene expression.
Cyclosporine has been shown to be effective in the treatment of immune-mediated
ocular diseases. However, it is associated with renal, hepatic, and neurologic
toxic effects that often limit its use in a high dose or for a long-term application.16-17 Sirolimus, a hydrophobic 31-membered
macrocyclic lactone (C51H79NO13), has been
demonstrated to inhibit a variety of experimental autoimmune diseases, retard
neoplastic growth, and prolong organ allograft survival.18-19
There is accumulating evidence that low doses of cyclosporine and sirolimus
have synergistic effects on immunosuppression that may significantly reduce
the risk of in vivo toxic effects.20-21
For all of these reasons, in this study we aimed to reevaluate the longevity
and intraocular safety of rAd-mediated gene delivery in real time after subretinal
injection, and to prolong transgene expression by transient immunosuppression
via combination of cyclosporine and sirolimus.
MATERIALS AND METHODS
GENERATION OF rAd
All manipulations regarding the rAd were performed in accordance with
the institutional biosafety guidelines. The GFP was selected as a reporter
gene because its expression can be monitored noninvasively in real time in
the eye.1-3 The
rAd, Ad.CMV.GFP, has an E1 and partial E3 region deletion, and a titer of
3.0 x 1010 plaque-forming units/mL was used in this study.
SUBRETINAL ADMINISTRATION OF Ad.CMV.GFP
All animal experiments adhered to the Association for Research in Vision
and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision
Research. Normal congenic pigmented and nonpigmented RCS/rdy+ rats
4 to 6 weeks of age were used in this experiment. A 2-µL solution containing
6.0 x 107 plaque-forming units of virus was delivered into
the subretinal space as described previously.2-3
Phosphate-buffered saline (PBS) was injected in the same way as a control.
Successful administration into the subretinal space was confirmed by the appearance
of a subretinal bleb under the operating microscope in nonpigmented eyes and
a partial retinal detachment by indirect ophthalmoscopy in pigmented eyes.
IMMUNOSUPPRESSION BY CYCLOSPORINE IN COMBINATION WITH SIROLIMUS
For immunosuppression, cyclosporine (Novartis Pharmaceuticals Australia
Pty Limited, North Ryde, Australia) was diluted with water for injection to
yield a concentration of 10 mg/mL. Sirolimus (kindly provided by Wyeth-Ayerst
Research, Monmouth Junction, NJ) was reconstituted in 0.2% carboxymethylcellulose
to a final concentration of 2 mg/mL. The subretinally injected rats were divided
into 4 groups: (1) vehicle, 0.2% carboxymethylcellulose (n = 9); (2) cyclosporine,
10 mg/kg per day (n = 7); (3) sirolimus, 2 mg/kg per day (n = 7); and (4)
combination of cyclosporine plus sirolimus, 10 and 2 mg/kg per day, respectively
(n = 11). Immunosuppression was initiated immediately after subretinal administration
of Ad.CMV.GFP by intraperitoneal injection once a day for 2 weeks. Carboxymethylcellulose,
the vehicle used to reconstitute sirolimus, was injected in the same way as
a control. All animals were housed in rooms that were suitable for work with
microorganisms and biological hazards under barrier conditions to prevent
infection. Animals were weighed weekly for 5 weeks to monitor their general
conditions under immunosuppression.
NONINVASIVE EVALUATION OF GFP EXPRESSION AND CLINICAL EXAMINATIONS
The GFP expression was observed by fundus fluorescent photography (FFP)
at 4, 7, and 10 days, then weekly for 10 weeks, as reported previously.2-3 The fluorescent signal was assessed
by 2 independent observers (W.-Y.S. and M.C.L.) and graded on a scale of 0
to 5 that involved both the transduced area and GFP signal intensity. For
area, a score of 5 indicated 80% to 100%; 4, 60% to 79%; 3, 40% to 59%; 2,
20% to 39%; 1, less than 19%; and 0, negative. For intensity, a score of 5
indicated very strong; 4, strong; 3, moderate; 2, weak; 1, very weak; and
0, negative. Fundus color photography was performed periodically in pigmented
eyes, and fluorescein angiography was performed when GFP expression was not
detectable.
RPE-CHOROID-SCLERAL FLAT MOUNT PREPARATION AND IMMUNOHISTOCHEMISTRY
After FFP, 8 eyes were enucleated at days 4, 7, 10, and 14 (2 eyes at
each time point) for RPE-choroid-scleral (R-C-S) preparation,2-3
and 4 eyes were enucleated and snap frozen in optimal cutting temperature
compound at day 7 for immunohistochemistry. Four PBS-injected eyes were enucleated
at 7 days as controls. The R-C-S whole mounts were examined by fluorescent
microscopy to evaluate the association between GFP expression and local cellular
infiltration. Frozen sections of 12 to 14 µm thickness were produced
for CD4+ and CD8+ cytotoxic T-lymphocyte immunostaining
as described previously,22 with the use of
monoclonal antibodies against rat CD4+ (1:50), CD8+
(1:50) lymphocytes, and CD68+ (1:500) macrophages (Serotec Inc,
Kidlington, Oxford, England).
ELECTRORETINOGRAPHY
Nine eyes received subretinal administration of Ad.CMV.GFP and were
treated with vehicle (n = 4) or cyclosporine plus sirolimus (n = 5), and 6
eyes from age-matched untreated rats were assessed by electroretinography
(ERG) at 4 weeks after injection. After anesthesia and pupil dilation, the
animals were allowed to adapt to the dark for 30 minutes before the scotopic
flash ERGs were performed. A platinum wire loop was placed on each cornea
to act as the recording electrode, and a reference electrode was connected
to the ear. Ground electrodes were attached to the animal's back. A xenon
strobe light placed 0.5 m in front of the animal presented the flash stimulus
at 0.25 Hz. Eight consecutive responses were amplified and averaged by means
of a bioamplifier/data recorder (MacLab/2e; ADInstruments, Sydney, Australia)
running Scope software (ADInstruments).
WHITE BLOOD CELL COUNTING AND SERUM BIOCHEMICAL ANALYSIS
By 5 weeks after injection, blood samples were taken from 20 rats under
immunosuppression. From each animal, 0.5 mL of blood was collected in a heparinized
tube for analysis of white blood cell count and differential count (Beckman
Coulter, Sydney, Australia). Another 2.0 mL of blood was collected for analysis
of glucose level and renal and liver function by standard procedures on an
automated analyzer (Hitachi 747 Autoanalyser; Boehringer Mannheim, Sydney,
Australia).
HISTOLOGIC EXAMINATION
Eyes were enucleated at 5 (n = 22) and 10 (n = 14) weeks after injection.
After eye enucleation and blood sample collection, the liver and kidney were
also harvested. The sampled tissues were fixed by 4% paraformaldehyde for
paraffin embedding. Paraffin sections 5 to 6 µm thick were processed
for hematoxylin-eosin staining to evaluate the retinal morphologic characteristics,
and sections from livers and kidneys were examined for the evidence of systemic
toxic effects of immunosuppression.
STATISTICAL ANALYSIS
All results are expressed as mean ± SD. Two unpaired groups were
compared with the 2-sample t test. Analysis of variance
was used to compare 3 or more groups. Differences were considered statistically
significant at P<.05.
RESULTS
TRANSGENE EXPRESSION AND CHANGES OF RETINAL PIGMENTATION
After subretinal injection of Ad.CMV.GFP, the retina images of nonpigmented
eyes appeared to be normal when observed by fundus color photography (Figure 1A). At 4 days after injection, strong
GFP expression was detected in all eyes, although there was some variability
in the size of the fluorescent area (Figure
1C). However, GFP expression rapidly declined to a basal level at
day 10 (Figure 1E) and was not detectable
at day 14 by FFP (Figure 1G).
|
|
|
|
Figure 1. Green fluorescent protein expression
and retinal images after subretinal injection of Ad.CMV.GFP at 4 days (A-D),
10 days (E), 2 weeks (G), and 4 weeks (F and H) after injection. A, B, and
F are retinal images obtained from fundus color photography. C to E and G
are images obtained from fundus fluorescent photography. H shows the disturbed
retinal pigmentation and choroidal capillaries of F, demonstrated by fluorescein
angiography. Arrows in B point to the route of needle penetration. Arrows
in F and H indicate the areas with disturbed retinal pigmentation. Asterisks
in B, F, and H indicate a site with progressive retinal depigmentation (original
magnification x6).
|
|
|
Considering the difficulty of distinguishing penetration-induced retinal
damage from rAd-induced toxic effects on histologic examination, fundus color
photography was performed in pigmented eyes (n = 8) to visualize the changes
after rAd administration. The pattern of GFP expression in pigmented eyes
was the same as that observed in nonpigmented eyes (Figure 1B, Figure 1D).
However, disturbed pigmentation was observed by 3 to 4 weeks after injection
(Figure 1F). Fluorescein angiography
further demonstrated an appearance of "RPE window defect" with stunning exposure
of the choroidal large vessels underneath the retina (Figure 1H). No vascular leakage was detected during fluorescein
angiography. The retinal depigmentation was not observed in PBS-injected eyes
(data not shown).
GFP EXPRESSION, LOCAL CELLULAR INFILTRATION, AND IDENTIFICATION
By examination of the R-C-S whole mounts, strong GFP expression was
observed at 4 days after injection while few inflammatory cells were detected,
and GFP was localized to the hexagonal RPE cells (Figure 2A, D). By 7 days, GFP expression was mostly detected in
the peripheral part of the virus-injected area, where the RPE morphologic
characteristics remained relatively normal. Infiltrating cells, however, were
observed in the center, accompanied by a decline in GFP expression. The RPE
cells became disorganized, resulting in the presence of "ghost" RPE cells
(Figure 2B, E). The GFP expression
was not detectable at day 14. Instead, a large amount of inflammatory cells
accumulated in the virus-contacted area, and RPE elimination was observed
(Figure 2C, F). Massive cell infiltration
and RPE damage were not observed in PBS-injected eyes (data not shown).
|
|
|
|
Figure 2. Photographs from fluorescent microscopy
of flat mounts of retinal pigment epithelium (RPE)–choroid–scleral
complex. A to C, Four, 7, and 14 days after injection, respectively. D, Hexagonal
RPE cells expressing green fluorescent protein. E and F, Cellular infiltration
and RPE damage in virus-contacted area at 7 (E) and 14 (F) days after injection.
Arrows in C point to the virus-contacted area. D, E, and F show higher magnifications
of the squares in A, B, and C, respectively (original magnification x10
[A-C], x50 [D-F]).
|
|
|
By fluorescent microscopy of cryosections, cellular infiltration was
observed in the subretinal space and neural retina from 7 days after injection
(Figure 3A). Immunophenotyping showed
that most of the infiltrating cells were CD8+ and CD4+
cytotoxic T lymphocytes (Figure 3B,
C), and macrophages (CD68+) were also detected (Figure 3D). By 4 weeks after injection, cell infiltration was dramatically
decreased, but retinal degeneration was observed in all rAd-injected eyes,
demonstrated as reduced number or total loss of the outer nuclear layer and
slightly affected inner nuclear layer (Figure
3E, F). Except for the occasional presence of macrophages (CD68+) at the site of injection, CD4+ and CD8+ cytotoxic
T lymphocytes and retinal degeneration were not observed in PBS-injected eyes
(data not shown).
|
|
|
|
Figure 3. Light micrographs showing cell
infiltration (A-D, serial sections) and retinal degeneration (E and F) after
subretinal delivery of Ad.CMV.GFP. A, Inflammatory cells in the choroid, subretinal
space, and neural retina (ch indicates choroid; pos, photoreceptor outer segments;
onl, outer nuclear layer; and inl, inner nuclear layer). B to D, Immunophenotyping
for CD8+ (B) and CD4+ (C) cytotoxic T lymphocytes and
CD68+ macrophages (D) at 7 days after injection. E and F, Micrographs
from a paraffin section showing retinal degeneration at an area between 2
arrows in E, 5 weeks after injection. F is a higher magnification of E (A-D,
stained or counterstained with hematoxylin, original magnification x100;
E, hematoxylin-eosin, original magnification x25; F, hematoxylin-eosin,
original magnification x100).
|
|
|
PROLONGED TRANSGENE EXPRESSION BY IMMUNOSUPPRESSION
Monitored by real-time observation, the efficiency and longevity of
GFP expression in vehicle-treated rats were similar to those observed in nontreated
rats. The GFP expression was detected in all groups at 4 days after injection
(Figure 4A-C and Figure 5). However, no fluorescent signal was detected in the vehicle-treated
group at week 2 (Figure 5). At 4
weeks after injection, the percentage of eyes with GFP expression was 67%,
50%, and 9% in groups treated with cyclosporine plus sirolimus, cyclosporine,
and sirolimus, respectively. Single drug application with sirolimus or cyclosporine
prolonged GFP expression for up to 4 and 9 weeks, respectively. By 10 weeks
after injection, 25% of the eyes in the cyclosporine plus sirolimus–treated
group still showed GFP expression (Figure
5).
|
|
|
|
Figure 4. Fluorescent images showing green
fluorescent protein expression in animals treated with transient immunosuppression
(A, D, and G were treated with cyclosporine and sirolimus; B, E, and H, with
cyclosporine; and C, F, and I, with sirolimus), 4 days (A, B, and C), 4 weeks
(F), 5 weeks (D, E, and I), 9 weeks (H), and 10 weeks (G) after injection
(original magnification x6).
|
|
|
|
|
|
|
Figure 5. Percentages of eyes with green
fluorescent protein (GFP) expression after recombinant adenovirus injection
and immunosuppression. The percentage of positive eyes includes any eye expressing
GFP signal detected by fundus fluorescent photography. The data represent
10 to 16 eyes per group from 4 days to 5 weeks, and 4 eyes per group from
6 to 10 weeks after injection. C indicates cyclosporine; S, sirolimus.
|
|
|
To compare the effect of different strategies on transgene expression,
the levels of GFP expression were graded by semiquantification that involved
both the area and intensity of fluorescent signal (Figure 6). Although cyclosporine and sirolimus, either alone or
in combination, prolonged GFP expression, GFP expression still decreased with
time at a slower rate during the first 2 weeks. Interestingly, a rebound GFP
expression was observed at 1 week after withdrawal of immunosuppression (3
weeks after injection) in animals treated with cyclosporine plus sirolimus
(P = .02) and cyclosporine (P
= .04) but not by sirolimus (P>.05). At 3 and 4 weeks
after injection, the levels of GFP expression in the sirolimus-treated group
were significantly lower when compared with the cyclosporine plus sirolimus–
and cyclosporine-treated groups, respectively (P<.01).
The GFP expression in cyclosporine plus sirolimus– and cyclosporine-treated
groups were comparable for up to 5 weeks. However, from 6 to 9 weeks, the
GFP levels in the cyclosporine-treated group were significantly lower than
those observed in the cyclosporine plus sirolimus–treated group (P<.05). Only the cyclosporine plus sirolimus–treated
group demonstrated GFP signal when observed by FFP at 10 weeks after injection.
|
|
|
|
Figure 6. Grades of green fluorescent protein
(GFP) expression in animals treated by different strategies of immunosuppression
after subretinal injection of recombinant adenovirus. C indicates cyclosporine;
S, sirolimus. Asterisk indicates P<.01 vs 2 weeks
after injection; dagger,P<.01 vs group treated
with cyclosporine and sirolimus; and double dagger, P<.05
vs group treated with cyclosporine and sirolimus; all by unpaired t test.
|
|
|
ATTENUATION OF rAd-INDUCED RETINAL DAMAGE BY COMBINED IMMUNOSUPPRESSION
The combined immunosuppression by cyclosporine plus sirolimus attenuated
but did not stop rAd-induced retinal damage (Figure 7). The ERG results are summarized in Table 1. By 4 weeks after injection, the implicit time of the a
and b waves and the mean amplitudes of the a wave were not significantly different
between groups. However, the mean amplitudes of the b wave significantly decreased
in the group treated with the vehicle (0.2% carboxymethylcellulose) (P = .03) and slightly decreased in the cyclosporine plus
sirolimus–treated group, but there was no statistical difference when
compared with the normal control group (P = .29).
|
|
|
|
Figure 7. Changes of retinal pigmentation
after subretinal injection of recombinant adenovirus, with or without transient
immunosuppression (A and B, cyclosporine plus sirolimus; C and D, vehicle),
4 weeks (A and C) and 8 weeks (B and D) after injection. Arrows indicate the
disturbed areas (original magnification x6).
|
|
|
|
|
|
|
Table 1. Dark-Adapted Electroretinography 4 Weeks After Subretinal
Injection of Ad.CMV.GFP With or Without Transient (2 Weeks) Immunosuppression
|
|
|
WHITE BLOOD CELL AND DIFFERENTIAL COUNTS AND ANIMAL TOLERANCE TO THE
IMMUNOSUPPRESSION
To monitor the effect of immunosuppression on systemic immunocompetence,
peripheral-blood samples were collected for white blood cell count and differential
count (Figure 8). Immunosuppression
by cyclosporine plus sirolimus significantly decreased the absolute number
of white blood cells, in comparison with the vehicle-treated group (P = .02). White blood cell differentiation showed that
the number of lymphocytes and monocytes was significantly reduced (P<.001 and P = .02, respectively). Cyclosporine
alone significantly reduced lymphocytes and monocytes (P = .02 and P = .03, respectively) but did
not affect the absolute number of total white blood cells (P = .09). Sirolimus decreased the number of lymphocytes (P = .02) but affected neither the number of monocytes nor the total
number of white blood cells (P = .85 and P = .43, respectively). In all 3 groups, neutrophils were not affected
(P>.30).
|
|
|
|
Figure 8. Peripheral white blood cell (WBC)
count and differential cell counts after transient (2 weeks) immunosuppression.
Blood samples were taken at 5 weeks. C indicates cyclosporine; S, sirolimus.
Asterisk indicates P<.05 vs vehicle treatment;
dagger,P<.01 vs vehicle treatment; double dagger,P<.01 vs group treated with cyclosporine and sirolimus;
and section mark,P<.05 vs group treated with cyclosporine
and sirolimus; all by unpaired t test.
|
|
|
Weight loss occurred in all animals treated with cyclosporine plus sirolimus
and cyclosporine, but not by sirolimus or vehicle (Figure 9). Cyclosporine plus sirolimus– and cyclosporine-treated
animals continuously experienced weight loss for 1 to 2 weeks but returned
to their baseline weight at 1 week after withdrawal of immunosuppression,
and all rats with initial weight loss gained weight beyond their pretreatment
levels at 2 weeks after the immunosuppression was suspended.
|
|
|
|
Figure 9. Variation in the weight of rats
measured at weekly intervals. The data represent 5 to 8 animals in each group
at every time point. The strategies of immunosuppression were performed by
daily intraperitoneal injection for 2 weeks. C indicates cyclosporine; S,
sirolimus.
|
|
|
Biochemical analysis showed that all groups had similar blood glucose
levels at 5 weeks after injection (Table
2). However, cyclosporine plus sirolimus and cyclosporine significantly
increased the levels of urea and creatinine and decreased the levels of inorganic
phosphate and alkaline phosphatase, but they did not change the levels of
alanine aminotransferase and aspartate aminotransferase. Except for a decreased
level of alkaline phosphatase, no other biochemical changes were found in
the sirolimus-treated group.
|
|
|
|
Table 2. Biochemical Analysis of Serum After 2 Weeks of Immunosuppression*
|
|
|
By histologic examination, no pathogenicity was found in the livers
and kidneys of all groups that had experienced transient immunosuppression
(data not shown).
COMMENT
A number of studies have examined rAd-mediated gene transfer after subretinal
injection, and the duration of transgene expression varies from 2 weeks to
several months.2, 4-5,7
In this study, the length of transgene (GFP) expression was monitored by FFP
in real time. We did observe a difference in method sensitivity between FFP
in living animals and fluorescent microscopy with R-C-S whole mounts. By fluorescent
microscopy, GFP expression was stronger than that observed by FFP, and in
some eyes, GFP expression occasionally lasted longer than observed by FFP.
In most eyes, however, we detected little GFP expression more than 2 weeks
after injection, indicating short-term rAd-mediated transgene expression.
The rAd-induced complications were demonstrated, as cellular infiltration
consisted of CD4+ and CD8+ cytotoxic T lymphocytes and
CD68+ macrophages, clumped retinal pigmentation, decreased b wave
of the ERG, and retinal degeneration on histologic examination. Finally, we
have dramatically improved transgene expression through temporary immunosuppression
by the combination of 2 synergistic immunosuppressants, cyclosporine and sirolimus.
Traditionally, several sites in the body have been described as having
immune privilege, including the eye. Earlier reports suggested that the subretinal
space is totally immune-privileged with respect to directly delivered rAd12, 23; however, recent investigations showed
dramatic controversy.14-15 In
this study, the gradual decrease of rAd-mediated transgene expression could
be the result of several changes, such as the cytomegalovirus promoter shutdown
and immune responses to the viral vector and GFP protein. We cannot exclude
that the reduction of GFP expression may be partially due to inactivation
of the cytomegalovirus promoter. However, we found a coincidence of reduction
of GFP expression with recruitment of inflammatory cells. Immunophenotyping
showed that most of the infiltrating cells are CD4+ and CD8+ cytotoxic T lymphocytes. We further demonstrated that combined immunosuppression
by cyclosporine plus sirolimus extended GFP expression by 6-fold. All of these
facts directly and indirectly indicate that the decreased GFP expression with
time is more likely due to immune responses. It has been recently demonstrated
that subretinal readministration of the recombinant adeno-associated virus
still resulted in GFP expression.24 In our
own hands, recombinant adeno-associated virus–mediated GFP expression
remained relatively consistent for up to 18 months.25
These facts indicate that the rAd-induced immune responses are related to
the presence of rAd vector rather than GFP protein. Previous studies have
demonstrated that the mechanisms involved in elimination of the rAd vector
by host responses include (1) innate immunity, (2) major histocompatibility
complex class I–restricted cytotoxic T-cell responses, and (3) types
1 and 2 helper T-cell–mediated cellular and humoral immune responses.26-27 The reason for the lost immune privilege
after subretinal delivery of rAd is unclear. It is highly possible that the
current technique of subretinal injection breaks the blood-retinal barrier
and allows some rAd particles to leak out of the eye. Once the immune system
is primed by the leaked rAd, the immune privilege of the eye is lost.14 Moreover, administration of a high dose of adenovirus
could also evoke a prominent delayed-type hypersensitivity after the second
administration of the same adenoviral vector.28
All these pitfalls limit the application of the first generation of rAd for
ocular gene therapy.
It is important to use generally accepted clinical techniques to evaluate
the intraocular safety when considering rAd for ocular gene therapy, particularly
by subretinal injection. In this study, fundus color photography clearly demonstrated
a progressive retinal depigmentation in the rAd-injected area. The results
of ERG, used to monitor the retinal function, have been reported previously
to be temporarily affected.13 The effect of
rAd on ERG response seems to be dependent on the rAd concentration. Delivery
of 6 x 104 plaque-forming units of rAd intravitreally or
subretinally resulted in a reversible decrease in ERG.13
In this study, increasing rAd concentration (6 x 107 plaque-forming
units) induced a significant decrease in the b-wave amplitude of the dark-adapted
ERG at 4 weeks after injection. Our results obtained from fundus color photography,
ERG, and histologic examination suggest that subretinal delivery of rAd affects
the neural retina function, indicating a potential retinal toxic effect.
The mechanism of retinal depigmentation is not clear. There is evidence
suggesting that activated peripheral cytotoxic T cells in response to rAd
migrate into the injected site, where they induce apoptosis through a Fas-Fas
ligand–dependent mechanism.29 Fas receptor
has been detected on the RPE cells, and binding of Fas-Fas ligand initiates
a cascade of apoptosis.30-31 This
theory may also explain our later results showing that the combined immunosuppression
improved GFP expression but was unable to stop the progress of retinal damage.
Other possible mechanisms by which cytotoxic T cells kill the RPE cells involve
perforin-granzyme and tumor necrosis factor interactions.32
It is also possible that the delivered rAd induces proinflammatory cytokine
release from RPE or photoreceptors and that they have pathologic effects even
before the appearance of inflammatory cells.33
Moreover, the rAd-induced RPE damage can further result in atrophy of the
choroidal capillary beds.34 All these alterations
will affect transportation of nutrients from the choroid to the neural retina,
which further leads to the irreversible retinal degeneration. Once the retina
is degenerated, the damage would be permanent.
Several approaches have been taken to circumvent the immune responses
to adenoviral vectors, such as deletion of several regions from the adenoviral
genome to engineer a less immunogenic vector. Unfortunately, further deletion
of the adenoviral genome makes the virus less efficient or more difficult
to propagate and obtain in high titer.35-36
The immune responses can be avoided provided that the vectors are delivered
into neonatal animals, which have an immature immune system,37
but this strategy is not of clinical relevance to ocular gene therapy.
Suppression of the host immune system by immunosuppressants such as
cyclosporine, tacrolimus, and etoposide has significantly improved the efficacy
of transgene expression.38-39
However, most of the currently available immunosuppressants display a narrow
range between efficacy and toxic side effects, making them less attractive
for clinical application.16-17
One strategy to overcome this limitation is to combine low doses of synergistic
drugs to achieve more therapeutic effect. In this study, we have demonstrated
that transient treatment with cyclosporine and sirolimus significantly prolongs
rAd-mediated transgene expression, and the combination is more effective than
single-drug application. From the results of white blood cell counting and
differential counting, we showed that cyclosporine and sirolimus, either alone
or in combination, dramatically decrease the number of lymphocytes and/or
monocytes in serum, with the combination being the most effective. These results
indicate that transient treatment with cyclosporine and sirolimus resulted
in systemically cellular immunosuppression that is sufficient to prolong transgene
expression.
Although we could not conclude whether the combined effect is synergistic
or additive, a number of previous studies have demonstrated that cyclosporine
plus sirolimus acts synergistically on immunosuppression.20-21
In the eye, cyclosporine and sirolimus inhibited retinal S-antigen–primed
lymphocyte proliferation, and they showed a marked synergistic effect over
a wide dose range as determined by a median-effect analysis.20
The synergistic effect of cyclosporine and sirolimus calculated in vitro was
further confirmed in vivo in the treatment of experimental autoimmune uveoretinitis,
demonstrated as complete inhibition of disease in all animals treated with
the combination regimen.20 The synergistic
effect of cyclosporine and sirolimus allows the reduced doses of each drug
to improve rAd-mediated transgene expression.
Interestingly, we observed rebound GFP expression in the groups treated
with cyclosporine plus sirolimus and with cyclosporine but not in the sirolimus-treated
group at 1 week after withdrawal of immunosuppression. Peripheral white blood
cell counts and differential counts suggest that strong immunosuppression
was achieved by cyclosporine plus sirolimus and by cyclosporine. It is possible
that the strong effect of cyclosporine plus sirolimus and cyclosporine during
the first 2 weeks not only circumvents the systemic immunity but also suppresses
cell activity. There is also another possibility, that continuous immunosuppression
by cyclosporine plus sirolimus and cyclosporine may inhibit the activity of
the cytomegalovirus promoter in certain contents, and withdrawal of immunosuppression
could relieve this inhibition.
Finally, some weight loss occurred after the combined immunosuppression,
but the weight was quickly regained after withdrawal of the treatment. Although
biochemical analysis of serum showed some changes in renal and liver function,
histologic examination did not support obvious damage.
AUTHOR INFORMATION
Accepted for publication February 1, 2001.
This study was supported by Meditech Research Ltd (Perth, Australia)
and the Dora Lush Postgraduate Research Scholarship (Australian National Health
and Medical Research Council, Canberra) (Dr Shen).
We thank Frank Shilton for fundus photography and Katrina Spilsbury,
PhD, for her valuable comments on the manuscript.
Corresponding author and reprints: Piroska E. Rakoczy, PhD, Department
of Molecular Ophthalmology, Lions Eye Institute, 2 Verdun St, Nedlands, Perth,
Western Australia 6009, Australia (e-mail: rakoczy{at}cyllene.uwa.edu.au).
From the Centre for Ophthalmology and Visual Science, University of
Western Australia, Perth (Drs Shen, Lai, Liu, Constable, and Rakoczy); Centre
for Pathology and Medical Research, Queen Elizabeth II Medical Centre, Perth
(Dr Beilby); and Vision, Touch & Hearing Research Center, Department of
Physiology and Pharmacology, University of Queensland, Brisbane, Australia
(Dr Barnett).
REFERENCES
| |
1. Bennett J, Duan DS, Engelhardt JF, Maguire AM. Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated
retinal transduction. Invest Ophthalmol Vis Sci. 1997;38:2857-2863.
FREE FULL TEXT
2. Lai CM, Shen WY, Constable IJ, Rakoczy PE. Preferential adenovirus-mediated transduction of cells at the sites
of laser photocoagulation in the rat eye. Curr Eye Res. 1999;19:411-417.
FULL TEXT
|
ISI
| PUBMED
3. Rolling F, Shen WY, Tabarias H, et al. Evaluation of adeno-associated virus-mediated gene transfer into the
rat retina by clinical fluorescence photography. Hum Gene Ther. 1999;10:641-648.
FULL TEXT
|
ISI
| PUBMED
4. Li TS, Adamian M, Roof DJ, et al. In vivo transfer of a reporter gene to the retina mediated by an adenoviral
vector. Invest Ophthalmol Vis Sci. 1994;35:2543-2549.
FREE FULL TEXT
5. Anglade E, Csaky KG. Recombinant adenovirus-mediated gene transfer into the adult rat retina. Curr Eye Res. 1998;17:316-321.
FULL TEXT
|
ISI
| PUBMED
6. Ali RR, Reichel MB, Hunt DM, Bhattacharya SS. Gene therapy for inherited retinal degeneration. Br J Ophthalmol. 1997;81:795-801.
FREE FULL TEXT
7. Reichel MB, Ali RR, Hunt DM, Bhattacharya SS. Gene therapy for retinal degeneration. Ophthalmic Res. 1997;29:261-268.
ISI
| PUBMED
8. Cayouette M, Behn D, Sendtner M, Lachapelle P, Gravel C. Intraocular gene transfer of ciliary neurotrophic factor prevents death
and increases responsiveness of rod photoreceptors in the retinal degeneration
slow mouse. J Neurosci. 1998;18:9282-9293.
FREE FULL TEXT
9. Dipolo A, Aigner LJ, Dunn RJ, Bray GM, Aguayo AJ. Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected
Muller cells temporarily rescues injured retinal ganglion cells. Proc Natl Acad Sci U S A. 1998;95:3978-3983.
FREE FULL TEXT
10. Dematteo RP, Markmann JF, Kozarsky KF, et al. Prolongation of adenoviral transgene expression in mouse liver by T
lymphocyte subset depletion. Gene Ther. 1996;3:4-12.
ISI
| PUBMED
11. Gagandeep S, Ott M, Sokhi RP, Gupta S. Rapid clearance of syngeneic transplanted hepatocytes following transduction
with E-1–deleted adenovirus indicates early host immune responses and
offers novel ways for studying viral vector, target cell and host interactions. Gene Ther. 1999;6:729-736. |
