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.
The authors thank Janice Lokken for preparing the tissue sections
and Inna Larsen for preparation of the manuscript.