Abstract
purpose. To determine the types of cells in the visual system of the mouse and
rat that can express a transgene delivered by an attenuated replication
competent Herpes simplex virus-1 (HSV-1) vector.
methods. C57/BL6 × BALB/C mice and Albino rats were treated with 1 ×
107 pfu of the HSV-1 ribonucleotide reductase mutant (hrR3)
expressing the Escherichia coli lacZ gene. The hrR3
virus was delivered by topical application to the cornea, intravitreal
(IV) injection, intracameral injection (IC), or stereotactic injection
into the visual cortex (VC). At specified times postinfection, animals
were killed and tissues were removed, fixed, sectioned, and stained
with X-gal or hematoxylin and eosin for histochemical and
histopathologic examination.
results. Topical delivery after corneal scarification in both mouse and rat
resulted in lacZ expression in 25% of the corneal
epithelial cells and 25% of the retinal pigment epithelium (RPE)
cells. Topical application without concurrent scarification also
resulted in transgene delivery to 20% of the RPE cells of the rat but
not the mouse. IV injection resulted in expression primarily in RPE
cells, with up to 100% of the cells expressing lacZ in
the mouse and rat. Other cells expressing the transgene included
ciliary body (CB) and optic nerve cells. Up to 25% of the retinal
ganglion cells in the rat expressed lacZ, but only
rarely in mice. IC delivery in rats resulted in expression in
trabecular meshwork, CB cells, RPE, and iris epithelium. Injection into
area 17 of the rat VC resulted in efficient labeling of the VC neurons
and neurons in the lateral geniculate nucleus without any evident
pathology or inflammation. Neither inflammation nor disease pathology
was observed in either the mouse or rat after any route of delivery.
conclusions. It was demonstrated that the hrR3 HSV-1 virus can deliver a
functioning gene to several cell types in the eye and neurons in the VC
and that the virus can move via retrograde transport to nuclei that
project to the VC.
Gene delivery to cells of the eye is a promising technology for
the treatment of a number of pathologic conditions. Viruses can
efficiently transfer genes to cells, and several viral based vector
systems have been used. Adenoviruses (AVs) have been widely used for
transgene delivery because of their ease of preparation and ability to
infect a wide range of cells. Delivery of recombinant AV to the eye via
subretinal injection results in reporter gene expression in the retinal
pigment epithelium (RPE) cells with limited expression in photoreceptor
cells at the site of injection.
1 2 3 4 Delivery via
intravitreal injections with AV vectors results in reporter gene
expression in retinal ganglion cells (RGCs), photoreceptor cells, and
RPE cells
3 5 6 and delivery via intracameral injection can
deliver a reporter gene to the trabecular meshwork, iris pigment
epithelium. and corneal endothelium.
7 8 Delivery of an AV
vector to the superior colliculus of mice also results in reporter gene
expression in the RGCs.
9 Therapeutically, recombinant AV
vectors have been used to deliver genes to the retina or optic nerve
after intravitreal and subretinal injections.
1 10 11 12 13 14 However, AV transgene delivery has been limited because of the host
immune response to viral expressed proteins.
15 16 17 Although the eye is a relatively immunologically protected site
(reviewed in
Ref. 18) intracameral injection of a recombinant AV in the
rabbit elicited a pathogenic immune response that led to tissue
destruction of the corneal endothelium in the anterior
chamber.
8 Similar results have also been reported in a
transfused human eye model.
19
Adeno-associated virus (AAV) has also been used to deliver transgenes
because of its ability to infect a wide range of human cell types and
lack of pathogenicity of wild-type virus. However, because of the small
genome size, AAV vectors are limited to delivery of less than 8 kB of
transgene, leaving little room for inclusion of large genes and
promoters or upstream regulatory elements. Gene delivery to the eye
using AAV via subretinal or intravitreal injection has resulted in
transgene delivery and expression in the RPE cells and to the
photoreceptors, RGCs, and Müller cells; however, the delivery was
limited to the area of the subretinal injection.
20 21 22 23 These subretinal injections also result in transient retinal detachment
that usually resolves within a few days; however, damage can persist at
the site of injection. In addition, AAV vectors have been used to
deliver therapeutic genes to photoreceptor cells and cells in the optic
nerve via subretinal
13 or intravitreal
24 injection.
Murine retroviral based vectors (mRV) have been used to deliver genes
to cells of the eye ex vivo
25 and in
vivo
26 27 ; however, the use of mRVs is limited because of
their inability to infect terminally differentiated cells. Recently,
human immunodeficiency virus (HIV)–based retroviral vector systems
have been used to deliver genes to the retina via subretinal
injection.
28 29 HIV vectors are able to efficiently
deliver a transgene to photoreceptor and RPE cells of neonatal rats;
however, delivery to cells in adult rats was much less efficient. In
addition, subretinal injections of the HIV-based vector produced a
local retinal detachment and resulted in infiltration of inflammatory
cells. Therapeutically, retroviral delivery of the HSV thymidine kinase
(TK) gene together with ganciclovir has been used to inhibit
proliferative vitreoretinopathy in a rabbit model.
27
Several herpes simplex virus (HSV) vectors have been developed for gene
delivery; however, to date, there are no published reports of their use
for gene delivery in the eye. The two most frequently used HSV-based
vectors include the following: replication defective HSV, with
deletions in immediate-early genes such as ICP0, ICP4, or
ICP27,
30 31 32 33 and amplicon vectors, which are deleted for
all essential HSV genes.
34 35 Amplicons require a helper
virus to complement HSV gene expression, and, like replication
defective viruses, require a helper cell line for propagation. Some
amplicon systems suffer from contamination by helper
virus,
36 and the production of high titer stocks with
amplicon vectors is difficult and requires multiple passages in vitro,
adding to the time of production. Because of the defective nature of
these vectors, they are also unlikely to cross synapses in the nervous
system.
One potential HSV-based vector system, which would avoid the use of
helper virus and helper cell lines, is a replication competent,
attenuated virus. Such viruses are deleted for an essential in vivo
virulence gene but retain the ability to be propagated in vitro. Thus,
these viruses can easily be grown and concentrated to high titer
(>10
9 pfu/ml), don’t require helper cell lines
or helper viruses; and because these viruses may retain limited
replication capacity in vivo, they may be able to cross neuronal
synapses. Limited replication by these vectors also may allow delivery
to adjacent cells, thereby expanding the number of cells expressing the
transgene. Studies with replication competent attenuated viruses with
deletions of the TK
37 or US3
38 gene showed
transgene expression when injected into the central nervous system
(CNS); however, this was accompanied by cytopathology at the site of
injection. Injection of a replication attenuated HSV-1 virus with a
deletion of the γ
134.5 gene caused severe
inflammation in the CNS.
39 Although these viruses have not
been tested in the eye, these results suggest that TK, US3, andγ
134.5 mutants may not be sufficiently
attenuated for use as gene delivery vectors.
The HSV ribonucleotide reductase (RR) gene is an important virulence
gene. The RR protein is composed of a heterodimer of the UL39 and UL40
gene products and functions to increase the cellular pool of dNTPs by
catalyzing the conversion of ribonucleotides to
deoxyribonucleotides.
40 Although HSV RR has been shown to
be nonessential in tissue culture, it is important for viral growth in
slowly growing and/or terminally differentiated
cells.
41 42 In addition we and others have shown that RR
null viruses are unable to cause disease after corneal or intravitreal
administration.
43 44 45 Ribonucleotide reductase mutants are
also impaired in their ability to cause death in mice after
intracerebral or intraperitoneal injection.
44 46 47 Moreover, although the virus can establish a latent state in sensory
nerve ganglia, RR mutants are unable to reactivate from
latency.
45 47 48 These findings suggest that an RR mutant
HSV-1 virus might be a useful vector for gene delivery to cells of the
visual system.
The work presented here shows that delivery of the RR mutant HSV-1
virus hrR3 to the rodent eye results in efficient gene delivery and
expression in the RPE cell layer regardless of the route of
administration. Surprisingly, delivery of the lacZ gene to
RPE cells was achieved by topical application to the intact cornea,
suggesting delivery via eye drops and patient self-administration
ration may be feasible. Efficient gene delivery and expression also
occurred in the ciliary body, trabecular meshwork, and corneal
epithelium. Other ocular cell types also expressed the lacZ transgene but at low efficiency. In particular, photoreceptors were
refractory to gene delivery. Injection of the hrR3 virus by various
routes into the mouse or rat eye showed no signs of ocular pathology
and little or no inflammation. In addition, injection of the hrR3 virus
into the visual cortex of rats resulted in lacZ expression
in neurons at the site of injection (area 17) and in the lateral
geniculate nucleus (LGN), which projects to the VC, with little or no
cytopathology or inflammation. These results demonstrate the
feasibility of using attenuated HSV vectors lacking RR for visual
system gene delivery.
Methods
Tissue Culture and Virus Preparation
African green monkey kidney cells (Vero) were grown in Dulbecco’s
modified Eagle’s medium supplemented with 5% serum (1:1 mixture of
fetal bovine serum and defined, supplemented calf serum), 100 U/ml
penicillin, and 100 μg/ml streptomycin sulfate as described
previously.
49 The construction of the hrR3 virus has been
described previously
41 ; briefly, the
E. coli
lacZ gene was cloned in frame with UL40 (RR large subunit) so that
expression of
lacZ is controlled by the UL40 promoter with
subsequent deletion of most of the UL40 gene reading frame. High titer
stocks of the virus were prepared on Vero cells as previously
described.
49 Virus stocks were purified for in vivo
delivery by high-speed centrifugation through a 36% sucrose cushion in
RSB (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl
2)
and then resuspended in phosphate-buffered saline (PBS).
50 Titers were determined by plaque assay on Vero cells.
In Vivo Gene Delivery
For ocular delivery of hrR3, C57/BL6 × BALB/C adult mice
(gift of Dr. Daniel Albert, University of Wisconsin, Madison, WI) and
albino adult rats (350 g; Harlan Sprague–Dawley, Indianapolis, IN)
were used. Mice were anesthetized with 5% halothane by inhalation and
the rats with intramuscular injection of xylazine (9 mg/kg) and
ketamine–HCl (90 mg/kg). Corneal scarification was performed as
previously described.
49 Briefly, the cornea was scarified
with a 30-gauge needle, and then 5 μl of the hrR3 virus suspension
containing 1 × 10
7 pfu in PBS was placed on
the cornea. Direct corneal delivery of the hrR3 virus was also
performed by placing a 5 μl drop of virus suspension containing
1 × 10
7 pfu directly on the cornea without
concurrent scarification. After virus delivery to the cornea, the
eyelid was held closed for 30 seconds, and the animals were returned to
their cages.
For intravitreal delivery, mice or rats were anesthetized as described
above, and a specified volume of the hrR3 virus in PBS was injected
into the vitreal cavity through a transcleral approach 2 mm posterior
to the cornea–sclera junction with a 30-gauge needle and Hamilton
syringe, taking care not to disturb the lens or the retina. The needle
was left in the eye for 10 seconds to allow ocular pressure to
stabilize. For intracameral administration, animals were anesthetized
as above and then injected with the hrR3 virus in PBS into the anterior
chamber via a transcorneal approach with a 30-gauge needle and Hamilton
syringe, taking care not to damage the lens or iris. All animal
procedures conformed to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research and NIH guidelines for responsible care
and use of animals and were approved by the institutional IACUC.
Tissue Processing
At specified times after gene delivery, the animals were killed by
cervical dislocation under halothane anesthesia for mice or by
CO
2 asphyxiation for rats. Eyes were removed and
prefixed in 10% formalin in PBS for 1 hour at room temperature and
then incubated in X-gal solution (1 mg/ml X-gal, 16 mM potassium
ferrocyanide, 16 mM potassium ferricyanide, 2 mg/ml
MgCl
2) for 48 hours as we described
previously.
51 The eyes were then postfixed in 2.5%
gluteraldehyde and 1% formaldehyde for 1 hour and then stored in a
solution of 1% formaldehyde and 6% sucrose in PBS at 4°C. The eyes
were then embedded in paraffin, sectioned (10-μm-thick) along
the pupil–optic nerve axis and counterstained with nuclear
fast red.
Quantification of X-gal Staining
Sections were observed microscopically, and tissue or cell types
were scored for the amount of X-gal staining as follows: 0 = 0%
of cells staining blue, 1 = less than 25% of cells staining blue,
2 = greater than 25% but less than 50% of cells staining blue,
3 = greater than 50% but less than 75% of cells staining blue,
and 4 = greater than 75% of the cells staining blue.
Inflammation and Ocular Pathology
Comparable sections were stained with hematoxylin–eosin and then
analyzed histopathologically. Briefly, inflammatory infiltrate was
scored as follows: 0, none; 1, inflammatory cells present on low-power
(40×) but less than 10 per field on highest power (400×); 2, 10 to
100 inflammatory cells per high-power field in the area of densest
infiltration; and 3, more than 100 inflammatory cells per high-power
field or epithelioid cell predominance. Tissue inflammation was scored
as follows: 0, none; 1, mild infiltrate; 2, marked infiltrate with
preservation of normal tissue structure; 3, marked infiltration with
some loss of tissue structure; and 4, obliteration of normal tissue
structure.
43 Scoring of the slides was done in a masked
manner with at least two scorers rating each infection route for each
animal.
Intracranial Injection
For delivery to the visual cortex, rats were anesthetized with
ketamine/xylazine as described above. Using aseptic precautions, a bone
flap was removed from the skull, and a unilateral injection was made in
the visual cortex (area 17
52 ). A 1-μl injection, 1 mm
deep into the cortex was made using a Hamilton syringe mounted on a
micromanipulator on a stereotax. A total of 2 ×
10
6 pfu of hrR3 was delivered over a 2-minute
period. Six rats were used; one received PBS, and five were given hrR3.
Three days later, the rats were deeply anesthetized with 60
mg/kg pentobarbitol sodium and perfused transcardially with 4%
paraformaldehyde and 0.25% gluteraldehyde. Brains were postfixed
overnight, and transverse sections (50-μm-thick) were cut on a
cryostat for X-gal staining and analysis.
Results
Gene Delivery in the Rat
Corneal Delivery.
After corneal scarification and topical delivery of the hrR3 virus, we
observed transgene expression in the corneal epithelium concentrated at
the sites of corneal scarification
(Table 1) . Expression of lacZ was also seen in cells resembling keratocytes in
the corneal stroma directly beneath the site of scarification (
Fig. 1A ). Occasional cells of the iris pigment epithelium, trabecular
meshwork, and the ciliary body also expressed the
lacZ transgene after corneal application. Surprisingly, the most efficient
gene delivery after corneal scarification was to the RPE with greater
than 25% of these cells displaying transgene expression. The
expression pattern was not uniform with greater numbers of
lacZ expressing cells near the ora serata and fewer near the
optic nerve. We also observed rare RGCs expressing the
lacZ transgene; however, these were few and randomly distributed.
Delivery of the hrR3 virus to the eye without prior scarification of
the cornea resulted in a similar pattern of
lacZ transgene
delivery
(Table 1) . We observed
lacZ gene expression in the
iris epithelium, ciliary body, and trabecular meshwork
(Fig. 1F) .
Transgene expression was observed in approximately 10% of the RPE
cells
(Fig. 1E) and in an occasional RGC, photoreceptor cell inner
segment, and cells of the optic nerve. Although,
lacZ transgene expression was distributed across the retina, greater
transgene expression was observed near the ora serata.
Intracameral Delivery.
After intracameral delivery with either 2 ×
10
6 pfu (1 μl) or 1 ×
10
7 (5 μl) pfu of the hrR3 virus, we observed
lacZ expression in 75% of the RPE cell layer
(Table 1) . A
few RGCs also expressed
lacZ, and these cells were randomly
distributed across the whole retina. In addition, intracameral delivery
resulted in transgene expression in 10% to 20% of stromal and
pigmented epithelial cells of the ciliary body and cells of the
trabecular meshwork as well as the corneal endothelium
(Fig. 1B) .
Intravitreal Delivery.
We delivered either 2 × 10
6 pfu (1 μl) or
1 × 10
7 (5 μl) pfu of the hrR3 virus to
the vitreal chamber. Regardless of the amount or volume of virus
delivered, we observed efficient labeling of the RPE, with nearly every
cell expressing the
lacZ transgene
(Table 1) . We also
observed
lacZ expression in approximately 25% of the RGCs,
which were spread throughout the retina
(Fig. 1C) . Rarely, we observed
lacZ expression in the inner nuclear layer, indicating
possible transgene delivery to horizontal, bipolar, or amacrine cells
(Fig. 1D) . In one eye, we observed a column of RPE cells, photoreceptor
cells, bipolar cells, and RGCs expressing the
lacZ transgene. Müller cells did not appear to express the transgene.
We also observed a few photoreceptor cells that expressed
lacZ in the cell body, but staining was not seen in the
outer segments. The
lacZ reaction product was also seen at
the tips of the photoreceptor outer segments where there was retinal
detachment during tissue processing, but this appeared to be due to RPE
material that had remained attached to the outer segments. Transgene
expression was also observed in cells of the optic nerve, which are
presumably glial cells. We also noted transgene expression in 10% to
20% of the iris pigment epithelium, trabecular meshwork, ciliary body
stroma, and pigmented epithelium as well as the corneal endothelium
after intravitreal virus delivery.
Pathology and Length of Transgene Expression
Parallel hematoxylin and eosin–stained sections from the rat were
examined for signs of inflammation or virus-induced pathology at 3, 7,
or 14 days postdelivery. We did not observe cellular infiltration in
the vitreal or anterior chambers, nor did we observe destruction of
tissue structure in the eye after any of the delivery routes even at 14
days postdelivery. Inflammatory responses were only seen when there was
obvious needle damage to the lens capsule during the injection
procedure. We did observe waning of the lacZ gene expression
beginning at 7 days, with little gene expression evident at 14 days
postdelivery.
Gene Delivery in the Mouse
Corneal Delivery.
Topical application of the hrR3 HSV-1 virus to the cornea after corneal
scarification of the mouse resulted in transgene expression similar to
that observed in the rat
(Table 2) . Expression of
lacZ was observed in the corneal epithelium
and underlying stroma as well as the RPE cells; however, in contrast to
corneal scarification delivery of hrR3 in the rat, we did not observe
lacZ expression in the iris pigmented epithelium, trabecular
meshwork, or ciliary body epithelium of the anterior chamber. Topical
delivery of the hrR3 virus to the mouse cornea without scarification
did not result in transgene expression in any cell type except for the
rare RPE cell
(Table 2) .
Intracameral Delivery.
After intracameral injection with 1 × 10
7 pfu of the hrR3 virus, the mouse eye displayed
lacZ transgene expression primarily in the ciliary body nonpigmented
epithelium (
Fig. 2C ) and the RPE cell layer; however, we did not observe transgene
delivery to other cells of the anterior chamber, nor did we observe any
RGC delivery.
Intravitreal Delivery.
Intravitreal injection of 1 × 10
7 pfu hrR3
virus in the mouse resulted in
lacZ expression in 100% of
the RPE cells
(Figs. 2A 2B) and occasional cells in the inner and
outer nuclear layers (
Fig. 2A , arrow).
Figure 2B shows a partially
tangential section of the retina, with extensive
lacZ expression in RPE cells showing the X-gal product distributed
throughout the cytoplasm of the RPE cells. In addition, intravitreal
delivery resulted in transgene delivery to the ciliary body
nonpigmented epithelium
(Fig. 2C) ; however, no other cells of the
anterior chamber expressed
lacZ after this route of delivery
in the mouse.
Pathology and Length of Transgene Expression.
Parallel sections of the eyes were also stained with hematoxylin and
eosin at 3, 7, and 14 days postdelivery so that we could observe
potential inflammation or pathology induced by the hrR3 HSV-1 virus.
Similar to our results in the rat, we did not observe cellular
infiltrate into the vitreal or anterior chambers, nor did we observe
destruction of tissue architecture or cytopathology in the eye after
any route of delivery even at 14 days postdelivery. As was observed
with the rat, lacZ gene expression began to decline at 7
days postdelivery, with little gene expression evident at 14 days
postdelivery.
Delivery to Visual Cortex
To assess transgene delivery to the visual cortex, the hrR3 virus
was stereotactically delivered to area 17 of the rat brain.
Representative results are shown in
Figure 3 . Expression of
lacZ occurred in numerous cells at the site
of delivery
(Figs. 3A and 3C) and extended approximately 1 mm on either
side of the injection track and 1 mm deeper in the visual cortex than
the tip of the needle used for delivery. Thus, the cells in a total
volume of 6.3 mm
3 were able to take up the vector
and express the transgene. The cells expressing
lacZ were
large and had characteristic neuronal morphology
(Fig. 3C) , including
some dendritic extensions that were positive for
lacZ (
Fig. 3C , arrow). Analysis of a control PBS-injected brain revealed
lacZ staining only in endothelial cells of blood vessels and
in the red nucleus as has been reported previously.
53 Thus,
lacZ expression in neurons in area 17 of the visual
cortex was due to virus-mediated transgene delivery.
To determine whether the virus could be transported to other regions of
the CNS, we also analyzed brain sections containing the LGN. As shown
in
Figure 3B , numerous LGN cells with the size and morphology of
neurons were positive for
lacZ expression in brains injected
with hrR3. Based on the volume of the LGN and thickness of the
sections,
54 we estimate that, on average, approximately
300 neurons in each LGN were able to express the
lacZ transgene. In contrast, no LGN
lacZ expression was seen in
PBS-injected brains, indicating that
lacZ expression was due
to the virally delivered gene. There was little, if any, evidence of an
inflammatory or glial cell reaction in areas where
lacZ was
expressed, and we saw no evidence for cytotoxic effects, suggesting
that the hrR3 virus is safe for use in CNS delivery. However, we should
note that only day 3 animals were evaluated, and this may be too early
to observe some pathologic responses.
36
Discussion
In this study, we examined transgene delivery and expression using
a replication competent HSV vector in the mouse and rat after corneal
scarification, intracameral injection, and intravitreal injection as
well as intracranial injection to the visual cortex in the rat.
Overall, the most prominent cell type expressing the transgene in the
eye was the RPE cell layer regardless of the route of delivery. Other
cells of the eye that expressed the lacZ transgene, although
to a much lesser extent, included the ganglion cells of the neural
retina, ciliary body, trabecular meshwork, and iris pigment epithelial
cells. Corneal delivery after scarification, although highly efficient,
was primarily restricted to the area damaged by scarification and
adjacent underlying keratocytes in the corneal stroma. Surprisingly,
corneal delivery without concurrent scarification also resulted in
transgene expression in several cell types of the anterior and vitreal
chambers (RPE in particular) in the rat but not in the mouse,
suggesting that specific species differences exist between ocular HSV
infections in the rat and mouse. In addition, in a few eyes of the rat,
intravitreal injection resulted in gene delivery and expression in an
occasional column of ganglion, bipolar, photoreceptor, and RPE cells.
As has been observed for other viruses inoculated into the eye, the
lens was refractory to infection by hrR3 by all infection routes. Even
when the lens capsule was accidentally damaged by the needle, the
exposed cells of the lens did not express lacZ, suggesting
that the lens cannot be efficiently infected by the hrR3 HSV-1 virus.
Intravitreal injections of AV and AAV appear to be much less efficient
at delivery to the neural retina than direct subretinal
injections.
3 4 6 14 21 With intravitreal delivery, AAV can
deliver genes to RPE cells and ganglion cells, whereas AV vectors can
efficiently deliver a gene to the iris pigment epithelium, corneal
epithelium, and RGCs with intravitreal delivery, but the RPE cells are
not efficiently transduced and those RPE cells expressing the transgene
are limited to the area surrounding the injection site. In our studies,
the attenuated hrR3 virus was capable of efficiently delivering the
lacZ gene to the RPE cells across the entire retina and was
able to deliver the transgene to up to 25% of the RGCs, but only
occasional cells of the inner nuclear layer and photoreceptor cells
after intravitreal injection. Because of the intense X-gal staining in
these sections, we were unable to determine the specific cells that
express the
lacZ transgene in the inner nuclear layer. In a
companion study examining hrR3 gene delivery in the monkey
eye,
51 we also found that intravitreal injection resulted
in efficient delivery of the
lacZ transgene to cells of the
RPE, suggesting that the efficient HSV-mediated RPE delivery is not
specific to just rodent species. Because the RPE cell layer provides
trophic support for the neural retina including the photoreceptor
cells, the RR mutant virus may be ideal for delivering
survival-promoting genes to the retina without the damage caused by
subretinal injections.
Gene delivery to trabecular meshwork or ciliary body cells may prove
useful in gene therapy strategies for glaucoma. Injection of
recombinant AV to the anterior chamber of mice results in gene delivery
to the corneal endothelium, iris pigmented epithelium, and trabecular
meshwork, with approximately 20% to 30% of the cells expressing the
delivered transgene.
6 Anterior chamber inoculation of AV
in rabbits also results in efficient delivery to the ciliary body and
other cell types, but this was accompanied by severe
inflammation.
8 10 We observed only limited transgene
expression in the anterior chamber in the mouse after intracameral
injection possibly due to technical difficulties related to injecting
into the restricted space; however, we did observe
lacZ gene
expression in the RPE cell layer. Nearly identical results were
observed in the rat, where intracameral delivery of the hrR3 virus
resulted in
lacZ expression in tissues of the anterior
chamber as well as the RPE cell layer. Recently, we showed that
intracameral injection of the hrR3 virus in the monkey eye resulted in
a similar pattern of
lacZ expression, with efficient
delivery of the
lacZ transgene to the ciliary body
epithelium and trabecular meshwork of the anterior chamber and also the
RPE cells, although delivery was accompanied by a transient
inflammatory response.
51 Thus, our results extend
trabecular meshwork and ciliary body gene delivery to the rat and
suggest that HSV vector systems may be useful for glaucoma-related gene
delivery.
We found that delivery of the hrR3 HSV-1 virus by corneal scarification
resulted in efficient gene delivery to the corneal epithelium only in
areas that had been damaged by the scarification. Topical application
of the hrR3 virus to the cornea without prior scarification did not
result in lacZ transgene expression in the corneal
epithelium or underlying keratocytes, indicating that damage to the
surface must be required for access of the virus to the corneal cells.
Corneal delivery of the AV and AAV do not appear to efficiently deliver
genes to the corneal cells in the absence of corneal injury. These
results suggest that restricted delivery to sites of corneal damage can
be achieved with the HSV-1 virus and may, thus, be useful for
modulating corneal wound healing.
There are a number of reports of intraocular gene delivery using AAV-,
AV-, and HIV-based vectors by subretinal
injection.
2 3 4 20 21 22 23 55 Injections by this route
typically deliver genes to the RPE cells and the photoreceptors, but
delivery is usually restricted to areas where retinal detachment
occurs. The most efficient gene delivery by this route appears to occur
either in young pup mice or adult mice undergoing photoreceptor
degeneration. These results suggest that the photoreceptor cells need
to be undergoing a growth or repair in order for efficient delivery to
occur. In addition, this delivery route resulted in localized retinal
detachment that, although resolving within a few days, did result in
damage to photoreceptor cells in the immediate area. For this reason,
we chose not to attempt subretinal injections in our studies.
The hrR3 virus was capable of delivering the
lacZ transgene
to cells of the retina regardless of the route of administration to the
eye. The route by which the vector virus reaches these cells is not
clear because the vitreal chamber was presumably not disturbed with
either the corneal delivery or the intracameral injection. Because
intracameral or intravitreal delivery of the hrR3 virus in a volume of
only 1 μl also resulted in efficient gene delivery to the retina, it
is unlikely that an increase in pressure resulting from the injection
forced the virus into the vitreous or the anterior chamber. We also
noted
lacZ expression in both chambers of the eye after
delivery of the hrR3 virus by corneal scarification and corneal drops,
neither of which should increase intraocular pressure. It is of
particular interest to note that topical delivery of the hrR3 virus to
the cornea of rats and mice could deliver the
lacZ transgene
to the RPE of both animals and the RGCs, ciliary body epithelium, and
trabecular meshwork of the rat without any disruption of the eye.
Scarification of the cornea before application of the virus enhanced
this transfer, but simply dropping the virus suspension onto the cornea
without prior scarification was sufficient to result in transgene
expression in these intraocular tissues. Liposome gene delivery has
been shown to deliver a transgene to the retina after topical
application to the cornea
56 ; however, no other report has
demonstrated vector-mediated gene delivery to the retina after corneal
application. Because the hrR3 virus is capable of delivering a
transgene to cells of the retina and angle without the need for
injection into the vitreous or anterior chamber of the eye, corneal
delivery allows for a noninvasive and presumably less traumatic
procedure for delivering transgenes to these cells. This may also
provide for gene delivery by self-administration by the patient, which
would be a considerable advantage.
Evaluation of gene delivery using reporter vectors depends on two
factors: the ability of the vector to enter the specific cell type and
the ability of the promoter to be expressed in a given cell. Thus, one
potential explanation for the lack of delivery to photoreceptors would
be a lack of expression of the marker gene. Therefore, in most gene
delivery studies, delivery may be underestimated. In our studies, the
lack of promoter expression as an explanation for poor delivery to
photoreceptors is unlikely for two reasons. First, we did observe rare
expression in cells in both the inner and outer nuclear layers. Second,
and more importantly, the lacZ gene was expressed from the
ICP6 viral promoter whose expression is enhanced by the virally encoded
ICP4, ICP0, and ICP27 genes, which are present in the hrR3 virus. Thus,
expression of the ICP6 lacZ marker gene is not entirely
dependant on cell-specific transcription factors in this vector system.
We, therefore, believe that the “poor” lacZ gene
expression is due to poor vector delivery and is probably related to a
lack of accessibility of the virus to the photoreceptors.
The eye is an immune-privileged site (reviewed in
Ref. 18) due to its
anatomic isolation as well as its unique immunomodulatory mechanisms,
and this may explain the lack of inflammation observed with the hrR3
virus as well as most other intraocular recombinant viral vector
infections. In fact, we did not observe immunologic cellular infiltrate
even 14 days postinfection, indicating that there is no immediate or
late response to the hrR3 virus in the eye. This is consistent with our
previous results showing that multiple intravitreal injections of an RR
null virus into the mouse eye does not cause immunologic pathology even
2 months postinfection.
43 The lack of an ocular immune
response to injection of other recombinant viruses is not uniform
because one study of AV showed that injection into the anterior chamber
of rabbits resulted in a severe inflammatory response in 3 of 8 animals
such that ciliary and iris epithelium and most corneal epithelium cells
were swollen and detached
8 ; however, this may be due to
species differences because most studies of virus-mediated gene
delivery to date have used rodent models.
Contrary to results in the rat and mouse, we observed inflammation and
cellular infiltrate after both intracameral and intravitreal injections
of the hrR3 HSV-1 virus in the monkey eye.
51 The
inflammatory response was characterized by an infiltration of
lymphocytes, polymorphonuclear cells, and macrophages into the anterior
and vitreal chambers; however, this inflammation appeared to be
transient and had waned by day 10 of the study. The immune response in
the monkeys was most severe in the virus-injected eyes but was also
seen in the control PBS-injected eye. One potential caveat to our
primate studies is that these monkeys had previously been used in other
ocular experiments, and it may be that the immune privilege of the eye
had already been compromised, contributing to a general inflammatory
response as a result of the needle injection alone. The inflammatory
response observed in the monkey may also reflect species differences in
response to the HSV virus and suggests that testing of recombinant
virus vectors in primates at least for pathology and toxicity is
essential because this model will presumably more closely mimic human
responses.
Injection of the hrR3 virus into area 17 of the rat visual cortex
resulted in efficient
lacZ expression at the site of
injection as well as at the LGN, which sends afferent projections to
the visual cortex. Although previous reports have described endogenous
lacZ expression and staining in the rat
brain,
53 the pH of our fixing and staining solution (pH
7.6) should have eliminated most of the background staining. In
addition, endogenous
lacZ expression was seen only in
endothelial cells and neurons in the red nucleus of PBS-injected rat
brain as observed previously
53 ; thus, the visual cortex
and LGN staining was due to the delivered transgene. Transgene
expression in the visual cortex and LGN appeared to occur in both
neuronal and nonneuronal cells, indicating that the hrR3 virus is
capable of infecting many different cells of the CNS as has been
described previously for other HSV vectors.
32 35 37 38 Delivery to the LGN likely resulted from anterograde transport of the
virus from LGN axonal termini located in the visual cortex. Transport
of virus with associated gene delivery has also been observed for CNS
injection of TK and ICP0 HSV null viruses.
36 57 Transport of the virus from the initial injection site may provide a
means for gene delivery not only to injected tissues but to innervating
tissues of the CNS as well. This phenomenon may be site-specific in the
brain; however, because delivery with HSV amplicon vectors can remain
localized in certain areas (see
Refs. 38 and
58 and Howard
Federoff, personal communication, May 1999). Previous investigators
have observed extensive tissue destruction and inflammation in the CNS
after injection of wild-type HSV
37 59 60 and even modest
tissue destruction at the site of injection with TK and ICP0 mutant HSV
vectors.
36 57 We did not observe virus-induced pathology
or immune cell infiltrate in any of the rats in our study 3 days after
injection of the RR mutant virus, suggesting that vectors attenuated by
mutations of RR may be more suitable for CNS delivery; however,
analysis of sections at 3 days after delivery may not have allowed
sufficient time for pathologic responses to become
manifest.
36
We also examined expression of the vector delivered lacZ gene 3, 7, and 14 days after vector delivery. Expression from the ICP6
promoter was strong at 3 days postinfection; however, 7 and 14 days
after vector delivery, expression began to decline in all transduced
tissues of the eye. Because the ICP6 promoter is an immediate
early/early promoter, we had not anticipated long-term expression from
this construct. The goal of this project was to identify the tissues of
the eye that were receptive to transgene delivery. In some clinical
situations, short-term delivery may be more desirable, such as
treatment of transient retinal ischemia or traumatic optic nerve
injury. However, promoters that express long term will need to be
identified before treatment of many eye diseases can be realized.
Previous investigations have examined the use of defective viral
vectors in the eye; however, these viruses have been limited in their
ability to efficiently deliver transgenes to cells across the whole eye
and have also been ineffective at delivering genes to the
photoreceptors except by the use of subretinal injection. One goal of
our study was to test the hypothesis that attenuated viruses may allow
for more efficient gene delivery to photoreceptor cells and neuronal
cells distant to the site of injection because of the possibility that
limited replication may allow the virus to cross synapses. We found
that the hrR3 HSV-1 virus can deliver a transgene to many cells of the
eye after corneal delivery, intracameral injection, and intravitreal
injection; however, we were not able to deliver genes efficiently to
the photoreceptor cells despite efficient delivery to the RPE cells,
which are in close contact to the photoreceptors. It is not clear why
photoreceptors are refractory to delivery, but possible explanations
include a lack of viral receptors on the outer segment membranes, a
lack of viral transport from the outer segments to the cell body, or
the presence of an extensive extracellular matrix surrounding the
photoreceptors. The success of gene delivery by subretinal injection
with concomitant retinal detachment suggests that access to the
photoreceptor cell body may be a critical factor.
Supported by a grant from the Retina Research Foundation (Houston, Texas; CRB). CRB is the Retina Research Foundation Alice McPherson Professor.
Submitted for publication June 30, 1999; revised November 30, 1999; accepted December 30, 1999.
Commercial relationships policy: N.
Corresponding author: Curtis R. Brandt, Department of Ophthalmology and Visual Sciences, 6630 Medical Sciences Center, University of Wisconsin Madison Medical School, 1300 University Avenue, Madison, WI 53706.
crbrandt@facstaff.wisc.edu
Table 1. Summary of Ocular Gene Delivery in the Rat
Table 1. Summary of Ocular Gene Delivery in the Rat
| Cell Type or Tissue | Route of Administration | | | |
| CD | CS | AC | VC |
| Corneal epithelium | 0 | 1 | 0 | 0 |
| Corneal endothelium | 0 | 0 | 1 | 1 |
| Iris pigment epithelium | + | 1 | 0 | 1 |
| Trabecular meshwork | + | 1 | 1 | 1 |
| Ciliary body | 1 | 1 | 1 | 1 |
| Lens | 0 | 0 | 0 | 0 |
| Ganglion cells | + | 1 | 1 | 2 |
| Photoreceptors | + | 0 | + | + |
| RPE | 1 | 2 | 3 | 4 |
| Optic nerve | + | 0 | 0 | 1 |
| Sclera | 1 | 1 | 1 | 1 |
Table 2. Summary of Ocular Gene Delivery in the Mouse
Table 2. Summary of Ocular Gene Delivery in the Mouse
| Cell Type or Tissue | Route of Administration | | | |
| CD | CS | AC | VC |
| Corneal epithelium | 0 | 2 | 0 | 0 |
| Corneal endothelium | 0 | 0 | 0 | 0 |
| Iris pigment epithelium | 0 | 0 | 0 | 0 |
| Trabecular meshwork | 0 | 0 | 0 | 0 |
| Ciliary body | 0 | 0 | 3 | 2 |
| Lens | 0 | 0 | 0 | 0 |
| Ganglion cells | 0 | 0 | 0 | 1 |
| Photoreceptors | 0 | 0 | 0 | + |
| RPE | + | 1 | 3 | 4 |
| Optic nerve | 0 | 0 | 0 | 1 |
| Sclera | 0 | 1 | 1 | 1 |
The authors thank Janice Lokken for preparing the tissue sections
and Inna Larsen for preparation of the manuscript.
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