Abstract
Gene
therapy of the eye has a high potential of becoming the preferred
treatment of a number of eye diseases. Because of its easy
accessibility, all the tissues of the eye can be reached and genetically
manipulated with nowadays standard gene delivery technologies. Gene
therapy offers the possibility to do both, correct a genetic defect by
replacing the mutated or missing gene and that of using genes as drugs.
Gene drugs would be more specific and would have a longer duration of
action and less toxicity than conventional drugs. Examples of both
applications are beginning to emerge. Using gene replacement, vision has
been restored in several patients of Leber congenital amaurosis (
Maguire et al., 2009). Some gene drugs, such as siRNA, are currently in clinical trials to silence angiogenic factors in macular de
1. Introduction and basis of gene therapy for glaucoma
Glaucoma
is an optic neuropathy caused by the degeneration of the retinal
ganglion cells (RGCs). Glaucoma occurs more frequently in the older
population and it is the second cause of worldwide blindness (
Quigley, 1996).
Blindness caused by glaucoma is irreversible. Currently, the only
treatment available for glaucoma is the administration of daily drops or
surgery. However, the use of daily drops is practically difficult and
quite cumbersome for an aging individual leading to a high degree of non
compliance. The search for an alternative treatment where the use of
the medication could be reduced to once or twice a year is highly
desirable.
Using genes as
drugs could bring us such possibility. Genes, or fragments of genetic
material, can be delivered inside a given cell and allowed to make their
encoded protein for long periods of time. Because the sequence of the
human genome is now complete, the DNA of every single enzyme or protein
can be isolated, cloned and amplified for an unlimited number of times.
Proper engineering of the molecule and insertion of regulatory elements
in a gene clone can theoretically control its expression and modulate
the targeting and abundance of the wanted product. Although multiple
limiting factors and interferences do still exist and detract from a
perfect outcome, advances in treatment of all diseases by gene therapy
have been staggering.
In
glaucoma, the major risk factor is elevated intraocular pressure (IOP).
Elevated IOP is the result of an increased resistance of the trabecular
meshwork to the flow of the aqueous humor as it exits the eye. The
trabecular meshwork is a sophisticated spongiform tissue formed by a
loose structure of cells and extracellular matrix (ECM) organized in
characteristic architecture. The tissue is bordered by a one-cell layer
which forms the inner wall of the Schlemm’s canal. Although every cell
layer of the trabecular meshwork could be causing dysfunction, it is
well established that the increased resistance in glaucoma is due in
great part to an accumulation of ECM and the formation of sheath-derived
plaque materials (SD-plaques) (
Lütjen-Drecoll, 1973 and
Lütjen-Drecoll et al., 1986).
For
the gene therapy treatment of glaucoma we could target either of the
two main tissues: the trabecular meshwork and the RGC cells. The goal of
targeting the trabecular meshwork will be to use genes to lower IOP
while the goal of targeting RGC will be that of using genes to protect
the cells from apoptosis and death (neuroprotection) (
Fig. 1).
Figure 1.
Target tissues for glaucoma gene drugs. Left: trabecular meshwork target; top: diagram of a meridional view section of the anterior segment of a human eye showing the routes of aqueous humor flow; bottom:
light microscopy photograph of a meridional section of a perfused
postmortem human anterior segment at the angle, showing the different
cellular regions of the trabecular meshwork and the Schlemm’s canal
(SC). Section stained with toluidine blue. Right: retinal ganglion cell (RGC) layer target; top: diagram of a meridional section of the human eye showing the delivery direction to the RGC; bottom:
light microscopy photograph of the human retina showing the different
cell layers and and emphasizing the RGC and nerve fiber layer which are
those affected by glaucoma. Section stained with eosin and hematoxylin.
The
correction of a genetic gene defect in glaucoma would also be possible.
However, this approach would most likely be limited to single severe
cases involving young patients. The mutation would have to be well
characterized, and the gene replacement would have first to be conducted
in an animal model exhibiting the same gene mutation and phenotype than
that of the patient.
2. Viral vectors and siRNA
2.1. Viral vectors to deliver genetic material to glaucoma-relevant tissues
There
are four gene therapy tools being studied for gene therapy of glaucoma,
three common viral vectors plus an short interfering RNA (siRNA) (
Fig. 2).
The viral vectors are adenoviruses (Ad), adeno-associated viruses (AAV)
and Lentiviruses. All of them have been stripped off their own
pathogenic genetic material and engineered to carry potential
therapeutic gene expression cassettes. The cassettes contain the gene to
be delivered (transgene), a promoter, and possibly gene regulatory
sequences.
Figure 2.
Gene therapy tools for the trabecular meshwork. Top three diagrams: genome of the three most common viral vectors being studied for gene delivery to the glaucoma targeting tissues. Bottom:
diagram of the mechanism of silencing a gene by short interfering RNA
(siRNA); small molecules of siRNA bind to the RISC protein and are
carried to the target RNA which gets degraded by the RISC nuclease. As a
consequence, the gene is silenced.
Ads
are double-stranded DNA vectors, have high tropism and efficiency for
the trabecular meshwork; and have high capacity in their genome to
incorporate relative large genes. In vivo, they induce an inflammatory
response at high concentrations and their duration of expression is
short, between 1 to 3 weeks in most animals (
Borrás et al., 2001 and
Kee et al., 2001).
Though probably not the final tool for gene therapy, Ads are of great
use for trabecular meshwork pre-clinical studies. Studies in our
laboratory have investigated Ads carrying potential candidate genes such
as dominant negative RhoA, caldesmon and matrix metallopeptidase 1
(MMP1). These genes were found to decrease trabecular meshwork outflow
resistance in organ culture (
Gabelt et al., 2006 and
Vittitow et al., 2002) and to reduce IOP in living animals (
Gerometta et al., 2010 and
Spiga and Borrás, 2010) (see below).
AAV
are single-stranded DNA vectors, have high tropism for the retina, and
have the best gene therapy safety record to date. Once into the cells,
AAV can express the transgene for up to five years after a single dose (
Rivera et al., 2005).
An AAV2 vector carrying the RPE65 gene is currently being used in
clinical trials to reverse congenital blindness caused by a retinal
pigmented epithelium cell defect (
Stein et al., 2011).
Numerous examples of AAV vectors carrying neuroprotective genes (such
as ciliary neurotrophic factor, CNTF or brain-derived neurotrophic
factor, BDNF) and injected intravitreally have shown to protect RGC from
rat hypertensive models (
Hellstrom and Harvey, 2011).
Very recently, an exciting study using systemic delivery of an AAV
vector carrying the pigment-derived epithelium factor (PEDF) transgene
was able to protect RGC death in the glaucomatous DBA/2J mouse (
Sullivan et al., 2011).
AAV
viral vectors though, do not transduce the trabecular meshwork due to
their inability to form a double stranded DNA upon entering the cell.
However, because of their high desirability for long-term and safe gene
therapy profile, a second generation AAV vector has been developed, the
self-complementary AAV (scAAV) which overrides the limiting step. A
single intracameral dose of scAAV.GFP in living monkeys resulted in
positive gene delivery to the trabecular meshwork with early onset and
lasting for at least two years (
Buie et al., 2010) (
Fig. 3). These encouraging findings are setting the way for safe gene targeting of the trabecular meshwork.
Figure 3.
Expression
of reporter transgene after single intracameral injection of scAAV.GFP.
Delivery of the reporter gene encoding green fluorescent protein (GFP)
to the anterior segment of the living rat (left) and living monkey (right)
after single injection of the viral vector. Images of rat frozen
meridional sections at different time points were taken under a
fluorescent microscope (left). In the monkeys (right),
stable and long-term expression was monitored noninvasively with
goniophotography; images were taken with a fundus camera: positive gene
transfer, represented by the presence of green cells occurred in living
animals. The expression of the transgene lasted for about three months
in rats and for over two years in monkeys. Contralateral eyes injected
with phosphate-buffered saline (PBS) were negative.
Lentiviruses
are single stranded RNA vectors derived from human or simian
immunodeficiency viruses. They integrate into the host genome and as a
result, retain the ability to express the transgene for a long time.
Lentiviruses transduce both the trabecular meshwork and the RGCs, and
these vectors carrying genes for cyclooxigenase-2 (COX-2) and
pigment-derived epithelium factor (PEDF) lowered IOP in cats and
protected death of RGC in rats, respectively (
Barraza et al., 2010 and
Miyazaki et al., 2011).
2.2. Silencing genes with short interfering RNA (siRNA)
Short interfering siRNA
s
are double stranded RNA molecules 21–24 nucleotides long. Inside the
cells siRNA interacts with a nuclease-containing multiprotein complex
called RISC (RNA-induced silencing complex). After binding to RISC, the
siRNA unwinds and pairs with its target mRNA; the RISC nuclease cleaves
the mRNA at the target site and this cleavage precipitates a full
degradation of the mRNA molecule which now is unable to translate and
produce the protein. siRNAs are thus a powerful tool to silence unwanted
genes and/or modulate expression of downstream cascades. In initial
experiments, the perfusion of post-mortem human anterior segments with a
fluorescent Cy3 labeled naked siRNA resulted in the entrance of the
molecule into the trabecular meshwork tissue. Subsequently, perfusion of
the siRNA for the glucocorticoid receptor (GR) did degrade its own RNA.
Furthermore, in the presence of dexamethasone (DEX), the GR siRNA
modulated also the expression of two DEX-induced genes (Myocilin and
Angiopoietin-like7). That is, silencing the receptor did control the
response of other genes induced by the glucocorticoid (
Comes and Borrás, 2007).
Continuation of these studies in living animals is showing positive
delivery of siRNA by intracameral injection to the trabecular meshwork
of rats (
Lawrence et al., 2011).
siRNA strategies are being developed by several pharmaceutical
companies. For glaucoma, a clinical trial study presented at the recent
ARVO meeting, showed reduction of IOP upon administration of eye drops
containing a naked siRNA to the β2 adrenergic receptors (
Ruz et al., 2011).
Studies coupling siRNA to different polymers and forming nanoparticles
are part of a big effort to increase efficiency of delivery of molecules
(
Huang and Liu, 2011).
3. Therapeutic gene targets and regulatory elements
In
parallel to optimizing delivery vehicles that will carry genes into the
cells, our investigations and those of others are being directed toward
the identification of gene targets which will lower IOP by modifying
the properties of the trabecular meshwork cells. Because the function of
any tissue is given by the expression of its genes, we first looked at
the most abundant genes expressed in the trabecular meshwork under
normal, physiological conditions. To understand which genes would be
relevant in the cause and response of the cells to glaucoma, we
performed similar studies subjecting the trabecular meshwork tissue to
glaucomatous insults, such as elevated pressure and glucocorticoids. For
a human model, we used the
perfused anterior segment perfusion
where tissues from donor eyes, up to 35 h post-mortem can be revived in
organ culture. Most important in such a model is the fact that the gene
response of a treated eye (OD) can be compared to that of paired
contralateral eye (OS) which has an identical genetic background. The
results are then not confounded by genetic differences among individuals
(
Fig. 4).
Figure 4.
Human anterior segment perfusion model. Top:
preparation of the postmortem human eyes for perfusion. Donor human
eyes, 30–40 h postmortem are bisected at the equator and have their
lens, iris and vitreous removed. The anterior segment is then mounted to
a custom-made 2-cannula polycarbonate dish and secured by an open ring.
Bottom: diagram of the perfusion system. The cultured chamber is maintained inside a CO2
incubator and perfused through one of the cannulas at constant flow
using a microinfusion syringe pump. The second cannula is connected to a
pressure transducer to monitor IOP. Gene drugs are injected through a
HPLC pump equipped with a 20-μl loop, that is intercalated between the
syringes and organ culture chambers. Pumps are controlled by a
custom-made computer program.
3.1. Identification of genes that respond to glaucomatous insults
Microarray
gene profiles performed with the RNA extracted from trabecular meshwork
of different individuals subjected to an elevated IOP insult (
Fig. 5) reveal a set of candidate genes that we termed the “IOP molecular biomarkers of the trabecular meshwork” (
Fig. 5) (
Comes and Borrás, 2009).
This set of genes has been cross-checked with other gene sets altered
by treatments with TGFβ2, DEX and with those which are present only in
the trabecular meshwork from glaucoma patients (
Borrás, 2008). The result of the cross-cross-check yielded a gene list representing a “molecular signature of glaucoma” (
Borrás, 2008).
An interesting finding from these studies is that genes altered by high
IOP could be divided into two subgroups, individual and general
responders (
Comes and Borrás, 2009).
This ability of some genes to respond differently to pressure in some
individuals provides the first molecular explanation to the different
IOP outcomes observed in the clinic. Interestingly, the 10 selected
genes identified in
Fig. 5
are representative of several cellular mechanisms that could affect
outflow facility. Angiopoietin-like 7 and matrix metallopeptidases 1 and
12 (MMP1 and MMP12) would affect the remodeling of the ECM; lysyl
oxidase and fibulin 5 would affect the collagen-elastin network; α-B
crystallin and myocilin would be involved in stress response; matrix Gla
would affect the calcification state of the cell; podoplanin and
chemokine CXCL2 would affect unknown pathways involved with the
lymphatic and immune systems.
Figure 5.
Identification
of pressure responder genes. (A) Diagram of the anterior segment
perfusion model during an elevated IOP experiment. To raise IOP, the
flow rate of one anterior segment is increased to achieve a ΔP
of ∼35 mmHg. The flow rate of the contralateral eye remains at baseline.
(B) Representative profile of a human eye pair perfused at elevated
pressure for three days. IOP is continuously monitored by a pressure
transducer and plotted with values obtained every 30 min. (C) Ten
selected pressure responder genes obtained from microarray profiles of
pressured insulted trabecular meshworks (Affymetrix).
3.2. Insertion of promoter elements to specify the site and extent of gene expression
Two
very important parameters to consider during the development of a gene
therapy regimen are tissue targeting and regulation of the expression of
the gene. To avoid unwanted, secondary effects due to gene-drug
expression in surrounding tissues, tissue-specific elements need to be
introduced in the gene promoter that would direct its expression only to
the wanted tissue. In addition, because gene-drugs could have a very
long duration of action (years), specific regulatory elements need to be
introduced in its promoter to be able to turn them on and off at will.
Ideally, a gene should be turned on “automatically”, in the presence of
the glaucomatous insult, and turned off when the insult is no longer
present. In some cases, those inducible elements are very well defined
in the literature as in the case of glucocorticoid response elements
(GRE, see below) (
Aranda and Pascual, 2001).
Other elements needed for our glaucoma application need to be
identified. A number of such vectors are currently in the pipeline. A
vector targeting the trabecular meshwork is carrying the promoter of α
B-crystallin driving what is called a reporter gene for easy detection
in histological sections. The strategy of this tissue-targeting vector
is based on a comprehensive review of the expression profile of genes
which are not expressed in tissues facing the anterior chamber, and in
the logistics that Ad vectors do not penetrate the lens capsule (where
αB-crystallin is highly expressed). A vector responding to
glucocorticoids is carrying the glucocorticoid response element (GRE) in
front of a basal promoter and driving the MMP1 potential therapeutic
gene. Such a vector has been fully characterized and proved to be
specific in an animal model of hypertension (see section 4). An
additional vector where the expression of the gene will depend of
whether elevated IOP is present, is also under development. Since no IOP
responding elements are yet defined, the beginning strategy has been to
use the promoter of a general pressure responder gene. The first
vectors contain the promoters of matrix Gla and angiopoietin-like 7
driving a reporter gene (secreted alkaline phosphatase) whose expression
can be measured biochemically along the time of high IOP exposure.
4. Application of gene therapy to treat steroid glaucoma
There
is a very good rationale as to why the development of a gene therapy
regimen for steroid glaucoma could result in an attractive and efficient
way to manage this type of ocular hypertension. Two percent of the
general population receives glucocorticoid treatments. Thirty to 40% of
the steroid-treated patients develop elevated IOP (
Armaly and Becker, 1965 and
Johnson, 1997).
At the cellular level, it is well established that steroid treatment
leads to an increase of ECM material which mimics that of primary angle
glaucoma (
Johnson et al., 1997).
This fact implies that several ECM targets could be used to counteract
the excessive build up. At the molecular biology level, the promoter
sequences that respond to glucocorticoids and turn on gene expression
have been very well defined in several other cells. Putting all this
information together led our laboratory to develop a strategy where GRE
elements would be inserted in front of an ECM degradation gene. To then
insert such a cassette into a gene therapy vector and to inject the
vector intracamerally into the eye of an animal model of ocular
hypertension. The intent of the project was to induce a reorganization
of the ECM only in the presence of the glucocorticoid. The final goal
was to reduce and prevent the steroid-induced elevated IOP.
4.1. Selection of matrix metallopeptidase 1 (MMP1) to degrade extracellular matrix (ECM) in the trabecular meshwork
Matrix
metallopetidases (MMPs) comprise a family of zinc-binding proteases
known to play a key role in the turnover of the ECM of the trabecular
meshwork (
Keller et al., 2009).
Previous studies on MMPs have shown that these enzymes are able to
increase aqueous humor outflow in perfused organ cultures (
Bradley et al., 1998).
A member of this family is MMP1, an interstitial collagenase which
breaks down collagens type I, II and III. Collagen type I is a main
component of the trabecular meshwork’s ECM scaffold. It constitutes the
central core of the trabecular meshwork beams. It was known that
treatment of trabecular meshwork with DEX in organ culture conditions
up-regulates collagen type I and down-regulates MMP1 (
Rozsa et al., 2006 and
Zhou et al., 1998). Further, treatment of human trabecular meshwork cells (HTM) with triamcinolone and prednisolone also down-regulates MMP1 (
Spiga and Borrás, 2010).
Therefore, MMP1 was selected as the gene target to investigate whether
its expression would counteract the steroid effect in living animals.
Overexpressing MMP1 under glucocorticoid conditions would serve not only
to counteracting its down regulation, but also would help reduce the
ECM accumulation.
4.2. Engineering of an inducible gene therapy vector to facilitate aqueous humor flow
An
adenoviral vector containing an inducible MMP1 cDNA was generated using
recombinant DNA techniques. The full coding MMP1 cDNA (1410 nt), able
to transcribe and translate a pre-active protein, was amplified from
laboratory HTM cells overexpressing MMP1. The basic promoter and GRE
sequences, able to induce a downstream gene, were extracted from a
commercial vector. Fusion of these two DNA fragments formed the
expression cassette which will be incorporated into the viral vector
Adh.GRE.MMP1. A second vector producing a mutated MMP1 protein was also
generated and used as negative control. This mutated MMP1 contained one
nt substitution at the catalytic binding site which leads to improper
folding and destroys its catalytic activity (Adh.GRE.mutMMP1) (
Fig. 6A) (
Spiga and Borrás, 2010).
Figure 6.
Generation
and assays of inducible gene therapy vectors for the treatment of
steroid-induced hypertension. (A) Schematic representation of
glucocorticoid-inducible viral vectors expressing recombinant MMP1.
Wild-type (active) and mutant vectors expressing MMP1 only after DEX
binding to the GRE sequences in the promoter. (B) DEX-induced
overproduction of recombinant MMP1 in HTM cells. Cells were infected
with wild-type and mutant viral vector, treated with 0.1 μM DEX and
harvested for RNA and protein. Levels of MMP1 RNA assayed by TaqMan PCR showed a very significant increase in the DEX treated cells compared to untreated controls (left). As well, levels of MMP1 protein assayed by western blots were highly increased in the treated cells (right).
(C) Enzymatic collagenase activity of secreted recombinant MMP1 in HTM
cells. Cells were infected and treated with DEX as in B and conditioned
media was harvested. Aliquots of the cell media were activated and
incubated with rat tail native collagen for 2 h. Samples were run on
gels and stained with Comassie blue to assay intact or degraded
collagen. Only activated samples obtained from DEX-treated cells
infected with the wild-type vector were able to break down collagen
(lane 2).
Response
of the potential vectors to glucocorticoids was extensively
characterized in primary cell and organ cultures conditions. As
expected, infection of the cultures with both vectors induced high
levels of MMP1 mRNA and protein (
Fig. 6B).
However, only the protein produced by the wild-type MMP1 had
collagenase activity and was able to degrade collagen. This important
property was tested in several assays. In a classical assay, purified
rat collagen was incubated with the activated conditioned media of cells
infected with MMP1 vectors and ran in polyacrylamide gels. Rat collagen
was seen degraded only in the gel lane which had been loaded with the
activated wild-type protein (
Fig. 6C).
A more sophisticated technology, state of the art assay, the
fluorescence resonance energy transfer (FRET), utilized an MMP substrate
peptide labeled with a fluorophore and a quencher. Cleavage of the
peptide with MMP1, which releases the fluorophore and is read in a
fluorophotometer, was observed only in the incubation of the
wild-type-infected conditioned media with the FRET peptide. Lastly, by
immunohistochemistry, double staining of the human perfused trabecular
meshwork with MMP1 and collagen type I antibodies revealed degradation
of collagen in the areas where levels of MMP1 were higher (
Spiga and Borrás, 2010).
Another
important property of the Adh.GRE.MMP1 vector was the fact that
overexpression of the MMP1 transgene could be synchronized with the
administration of DEX. Upon infection of HTM cells with the vector, MMP1
expression was consecutively on and off coinciding with the treatment
and withdrawal of the glucocorticoid. For a gene therapy drug
application, this characteristic is of upmost importance. Degradation of
the ECM in steroid-induced hypertension will occur only in the presence
of the steroid.
Altogether
the generated vector Adh.GRE.MMP1 seemed to contain all characteristics
required for its application to an steroid-induced in vivo model.
4.3. Sheep model of steroid-induced ocular hypertension
The
laboratories of Drs. Oscar Candia (Mount Sinai School of Medicine) and
Rosana Gerometta (Universidad Nacional del Nordeste, Corrientes
Argentina) have recently developed a model of glucocorticoid induced
ocular hypertension in Corriedale sheep (
Ovis aries) (
Gerometta et al., 2009).
Topical application of two drops of 0.5% prednisolone acetate
(Ultracorteno, Novartis) 3× daily into one eye of the sheep results in a
∼2.5× IOP increase within 1–2 weeks. IOP values, read with a Perkins
applanation tonometer, are between 9–11 mmHg at baseline and raise to
25–28 mmHg. Pressures of the contralateral eye, receiving artificial
tears, remain at baseline levels. In contrast to humans, this elevation
of IOP occurs in 100% of the glucocorticoid treated animals. The
elevated IOP is maintained during a 4 week application of the
prednisolone drops, persists after discontinuation of the treatment, and
returns to baseline levels over the course of one to three weeks (
Gerometta et al., 2009).
An
elevation of IOP is also obtained by a single sub-Tenon injection of
triamcinolone. One milliliter injection of triamcinolone acetonide
(40 mg/ml, Bristol-Myers Squibb) is administered under topical
anesthesia using a 30G needle. The injection is performed to create a
juxta-sclera depot rather that an intra Tenon injection (
Gerometta et al., 2010). Pressures of the injected eye are increased ∼2× at day 4 post-injection and remain high for about 2–3 weeks.
The
docile nature of these animals together with their 100% response to the
steroid makes the sheep model ideal for studies of gene therapy
treatment of steroid-induced hypertension.
4.4. Lowering IOP after glucocorticoid administration with an inducible viral vector
In
a study involving a total of six sheep, baseline pressures were taken
for one week and prednisolone was administered daily afterward in both
eyes. At four days post-prednisolone treatment, when the pressures had
risen to ∼2× baseline levels, one eye of each sheep received an
intracameral injection of 1 of 3 adenoviral vectors while the
contralateral eyes remained uninjected. One vector carried the wild-type
MMP1 (Adh.GRE.MMP1) (
Fig. 7A), the second vector carried the mutated form of the protein lacking the active catalytic site (Adh.GRE.mutMMP1) (
Fig. 7B),
and the third vector was “empty”, that is, carrying no transgene
(Ad.Null). The contralateral eyes were left uninjected. The IOP stayed
elevated in all eyes that were either uninjected or injected with the
control vectors for the duration of the prednisolone instillation
However, in eyes injected with the active MMP1 the elevated IOP returned
to the lower baseline values two days after the viral injection (
Gerometta et al., 2010).
Low pressures of the wild-type injected eyes persisted for
approximately 10–15 days longer, which correspond with the published
data of the duration of the expression of adenoviruses in living animals
(
Fig. 7) (
Borrás et al., 2001 and
Kee et al., 2001).
Figure 7.
Adh.GRE.MMP1
injection lowers steroid-induced hypertension in sheep. Representative
corticosteroid regimen and IOP values from two sheep: (A) sheep injected
in one eye with the active Adh.GRE.MMP1. (B) Sheep injected in one eye
with the inactive mutant Adh.GRE.mutMMP1 (B). Contralateral eyes also
treated with corticosteroid, remained uninjected. (C) Representative
plots of the IOP measurements. Only the eye injected with the active
gene therapy vector experimented a decrease from the elevated IOP
induced by steroids.
An
additional four sheep received sub-Tenon injections of triamcinolone in
both eyes. Four days later, IOP increased from 11.3 ± 0.3 to
22. ± 0.8 mmHg in one eye and, from 9.7 ± 0.2 to 22.1 ± 0.2 mmHg in its
contralateral. One eye of each sheep was then injected with the
wild-type Adh.GRE.MMP1 while the paired eye was injected with the vector
carrying the mutated form of the protein (Adh.GRE.mutMMP1). Two to
three days after viral injections, IOP was significantly reduced in the
eyes injected with the active MMP1 (11.6 ± 0.4) but remained elevated in
the eyes injected with vector carrying the mutant protein which lacks
the catalytic site (22.1 ± 0.2 mmHg). However, in contrast with the
prednisolone experiments, where the reduction was maintained for almost
two weeks, the reduction observed with the wild-type vector lasted just
for three to four days (
Gerometta et al., 2010).
The causes of the short reduction after triamcinolone injection are not
known and need further investigation. Perhaps, variability in the
formation of the depot and release of the steroid plays a role in
overriding the effect of the vector.
None
of the sheep eyes injected with any of the adenoviral vectors showed
adverse clinical signs. There were no signs of hyperemia or
inflammation, and the corneas remained clear. This first case of
lowering elevated IOP by a gene therapy vector in a large animal
provides the initial steps for the development of a new avenue of
treatment for ocular hypertension induced by steroids.
4.5. Prevention of steroid-induced elevated pressure by pre-injection with an inducible viral vector
In
addition to lowering the steroid-induced IOP, the adenoviral vector
which delivers the active MMP1 is also able to prevent the
steroid-induced IOP elevation. In two independent experiments,
Adh.GRE.MMP1 was injected at day 0 and prednisolone instillation started
two or three days after injection. Pressure elevation did not occur
until 12 to 15 days post-viral injections, in concordance with the
duration of expression of the transgene in an adenoviral vector (
Fig. 8).
Figure 8.
Prevention
of steroid-induced hypertension. Pre-injection of the active gene
therapy vector Adh.GRE.MMP1 prevents the elevated IOP induced by the
corticosteroid. Contralateral uninjected eye received two corticosteroid
treatments and induced elevated IOP.
Similarly,
the same Adh.GRE.MMP1 vector was able to avoid the doubling pressure
increase induced by the triamcinolone. At day 0, with baselines
pressures of 9.4 ± 0.1 mmHg, the eyes of two sheep were injected with
the active viral vectors followed by triamcinolone injections one day
later. Pressures remained close to baseline levels for four days,
reached a mid-value of 12.9 ± 0.5 mmHg at five days and attained the
expected triamcinolone doubling response of 21.3 ± 0.3 mmHg at 11 days.
These findings indicate that during the time the virus is producing the
therapeutic protein (10–15 days), there is a counteractive, protective
action that impedes the steroid to exert its full potential.
5. Conclusion and future directions
During
the past few years, there have been important advances in the use of
gene therapy for the treatment of eye diseases. Without a doubt, the
most important success has been the restoration of vision by the
replacement of the RPE65 gene to patients of Leber congenital amaurosis (
Stein et al., 2011).
In addition of demonstrating efficacy by delivering the wild-type gene,
these clinical trials have served to validate the safety of the AAV
viral vector for the use of gene therapy in the human eye. The
importance of this safety issue cannot be underestimated. Regarding
glaucoma, we have made considerable preclinical advances in all four
relevant fronts: potential therapeutic genes with regulatory elements,
safe and efficient delivery vectors, animal models, and significant
improvements in animal evaluation technology (from physiology to
imaging). There are now a considerable number of genes with
neuroprotective properties under study in several laboratories. These
genes are being tested in AAV vectors, which transduce RGC cells very
efficiently. There are also, as we saw above, potentially therapeutic
genes controlled by regulatory elements that could target the trabecular
meshwork and lower elevated IOP. We do have a second generation AAV
vector to transduce the outflow tissue and we are beginning to have good
animal models to test their efficiency. An important body of work is
ahead and we are not yet ready. However, the general outlook is that the
gene therapy approach for glaucoma looks much more feasible now than
what it did five years ago. We can only envision that the next five
years will be as productive and will take us to the trial of the first
gene therapy drug for glaucoma in the clinical setting.
