PEER REVIEWED
Gene Therapy for Age-Related
Macular Degeneration
DAVID KEEGAN, MD, PhD &
LUCIAN V. DEL PRIORE, MD, PhD
In its simplest form, gene therapy
involves replacing a defective or absent gene. This approach is being used for
the treatment of Leber's congenital amaurosis, which is due to a defect in the
RPE65 gene. However, this simple gene replacement approach is not likely to be
useful for the treatment of age-related macular degeneration (AMD), since this
multifactorial disease is unlikely to be due to
simple absence of a protein.
Current and future
technologies allow elegant manipulation of the physiology of any
micro-environment of the retina including the subretinal space. Gene therapy can
be achieved by targeted cellular transfection, manipulation of inducible gene
switches, the introduction of ribozyme technology that destroys or inactivates
mutant proteins, and/or ex-vivo cellular manipulation with subsequent tissue
transfer of transfected cells.
The purpose of this article is to provide an
understanding of some of these concepts, with an appreciation of how these
therapies may translate into the management of advanced AMD in the future. This
is not an exhaustive review and the reader is directed to more complete reviews
available in the recent literature.
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Figure 1.
Intravitreal and subretinal gene-therapy delivery.
A. Intravitreal injection of vector.
B. Subretinal
vector injection.
C. With time, the virus vector infects the RPE cells and
the transmitted gene inserts into the host genome.
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BASIC MECHANISMS OF GENE THERAPY
The basic strategies of gene therapy can be
separated into gene replacement, gene silencing, or delivery of a gene to
produce a protein locally. These genes must be delivered in such a way as to
introduce the gene into the cellular genome such that the local effect may be
elicited.
■Replacing Absent or Defective
Gene/Protein.
The simplest application of gene therapy occurs when patients develop a disease
due to complete absence of a protein product. Typically, this occurs in
autosomal recessive diseases in which no protein is made, and absence of the
protein leads to disease. The concept here is that a viral vector can be
engineered to carry DNA that codes for the missing gene; the DNA is then
inserted into the host DNA or maintained as a plasmid within the host nucleus
such that transcription of healthy protein will occur. Several factors can be
used to ensure the specificity of targeted gene delivery, including targeted
vector delivery to a specific anatomic location, such as the vitreous cavity or
subretinal space (Figure 1); the use of a cell-specific promoter region on the
gene construct to limit gene expression to certain cells types (eg, using a
rhodopsin promoter to limit gene expression to photoreceptors as a means of
targeting these cells for photoreceptor-based treatments); use of viral vectors
that have a high affinity for a particular cell type; or performing gene therapy
ex vivo in explanted cells in culture, which can then be introduced into the
subretinal space or vitreous cavity. In principle, introduction of a specific
local source of protein production should be therapeutic if the disease is due
to protein absence; the poster child for treatment of autosomal recessive
retinal disorders is represented by recent groundbreaking pre-clinical studies
of gene therapy in a dog model of Leber's Amaurosis (RPE65-/-).1,2
The role of gene replacement therapy in the treatment of AMD is not known at the
current time, but it is unlikely that this
disease is due to absence of a
single protein.
■Gene
Silencing. This approach is favored for the treatment of disorders that
are due to the presence of a mutant or aberrant protein; normally these
disorders are present in diseases that are dominantly inherited. Typically, a
normal protein product is present as well due to expression of a normal gene on
the other allele, but disease is caused by abnormal protein caused by expression
of an abnormal gene. Here the goal of treatment is to destroy or remove the
abnormal protein. This can be achieved using RNA inhibition (RNAi and siRNA) or
ribozyme technology; where these agents are capable of binding to a cleavage
site of the target RNA resulting in disruption of its transcription and
expression (Figure 2). Ribozymes or siRNAs can be delivered with viral or
liposomal vectors.
■Enhanced Production of Beneficial Protein.
Another potential application of gene therapy is to introduce a gene that will
result in an increase in the local concentration of a beneficial protein. Here,
gene therapy would allow continuous expression of a protein that could be used
to treat the disease. The best example of this approach in AMD is the recently
reported phase 1 study using Adenoviral-vector delivered PEDF (AdPEDF.11) to
suppress choroidal neovascularization in AMD.3 Other examples include
the production of a soluble VEGF receptor (Flt-1)4 or delivery of
growth factors in animal models of inherited retinal disease.5,6
GENE REGULATION
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Figure
2. RNA interference using inhibitors of RNA (siRNA and
ribozymes).
A. The therapeutic siRNA is delivered and enters the
cell by endocytosis in a vector or liposomal system. The
siRNA binds to host RNA-induced silencing complex (RISC)
in the cell. This complex loses a strand from the siRNA,
which then binds with host cell messenger RNA. This
cleaves the host mRNA and prevents protein production.
B. Ribozymes are
small RNA molecules, generally of a "hammerhead" or
"hairpin" configuration. They bind to specific cleavage
sites on host messenger RNA and make them more
susceptible to intracellular RNA hydrolases. Messenger
RNA is cleaved and protein translation does not occur. |
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In order for gene therapy to be safe and
effective, it is important that techniques are developed to control the
expression and behavior of the gene following insertion. Simple introduction of
a therapeutic gene may not be sufficient in the long run if the protein product
is produced at concentrations that are either too low or too high for the
desired therapeutic effect. Careful regulation of gene expression, and therefore
the levels of gene product, can be achieved using a relevant promoter element on
the gene to deliver protein to the target tissue. Examples of this approach
include the use of rhodopsin promoters in the photoreceptors, or PEDF promoters
in the retinal pigment epithelium (RPE) and choroid, to regulate gene
expression. Altered gene expression can also be secondary to changes in the
cellular microenvironment such as hypoxia; here it is possible to use
hypoxia-sensitive response elements (HRE) to regulate the expression of anti-angiogenic
genes (Figure 3).
Target gene expression may be modified further by
using a switch system chemosensitive to a tetracycline (eg, doxycycline); in the
Tet-ON system the gene of interest is linked to a tetracycline response element
(TRE) which in turn is modified by a reverse tetracycline-dependant
transactivator (rtTA: Figure 4). Doxycycline binds to rtTA which in turn binds
to and activates the TRE inducing upregulation of the target gene. The Tet-OFF
system is mediated through the tetracycline-dependant transactivator (tTA),
where the increase in gene activity occurs following removal of doxycycline.
Unbound tTA binds to the TRE inducing gene expression.
The choice of gene
delivery system can also aid the regulation of gene expression. Adenoviral
vectors do not maintain long-term expression well as they do not integrate into
the host genome, and thus they are ideal for acute rather than chronic disease.
In contrast, adeno-associated viruses can maintain long-term expression due to
stable host genome integration.
GENE THERAPY VECTORS
Gene therapy requires an efficient delivery
system. Several factors control the efficiency of gene therapy and subsequent
protein production:
■Gene insertion in target tissue.
This is particularly important in relation to the retina, as transduction of
neural retina is more difficult than RPE. This can be overcome by the choice of
vector systems, since some viruses have a predilection for transfection of
different target tissues. For example, AAV vectors can achieve good
photoreceptor and RPE transduction whereas retroviral vectors only transfect
dividing cells and may not be appropriate for retinal gene therapy (Table 1).
■Appropriate passenger genetic material (eg,
switches or tissue/physiological specific promoters). See details of the
Tet-ON or Tet-OFF system above and in Figure 4. Physiological promoters
represent another attractive way of regulating local protein production. For
example, in principle one can fine-tune the level of VEGF expression to the
amount of VEGF by tying the expression levels of anti-VEGF agents to the
presence of an upstream modulator of VEGF production, such as the HRE which
controls VEGF production.7 An HRE responsive promoter sequence can be
placed on the delivered gene, resulting in an anti-VEGF protein such as Flt-1,
the soluble VEGF receptor, being produced. This type of system effectively
creates a local auto-regulatory pathway (Figure 3).
Viral Vectors
Modified viruses are the most common vectors used
in gene therapy as they allow effective intracellular delivery of genetic
material. Several factors are important in determining the ideal viral vector
for a certain therapeutic use, including the predilection of the viruses for
certain tissues and cells, and the size of the DNA fragment that can be inserted
with a given vector (eg, adeno-associated virus and photoreceptors).8
Potential viral vectors for ocular use include adenovirus, adeno associated
virus, retroviruses, and lentiviruses.
Ex
vivo Gene Therapy
Ex vivo gene therapy, in which explanted cells
are transfected prior to reimplantation in the eye, can provide an attractive
treatment alternative for patients with age-related macular degeneration. In
principle, RPE modified ex vivo can be delivered into the subretinal space to
provide physiological levels of key molecules, such as anti-VEGF agents, PEDF,
growth factors such as ciliary derived neurotrophic factors (CNTF), or
anti-apoptotic agents including bcl-2
or caspase inhibitors. Caspases are
crucial enzymes in the apoptotic cascade and form an integral part of the final
pathway in the majority of causes of apoptosis. Caspase inhibition has been
shown to limit apoptosis in many cell systems including those subjected to
oxidative stress.9,10
The benefit of ex vivo gene therapy is the
potential to accurately determine the protein production of these cells prior to
inserting them into the eye. Disadvantages include difficulty sourcing cells for
transplantation, the potential for immune rejection of the transplanted cells,11-13
and alterations in protein production that can develop after the cells are
transplanted back into the eye.
Delivery of Vector
Delivery of the vector is an important
consideration in gene therapy. The issues to be considered include safety,
efficacy, reproducibility, and convenience. When considering vector delivery for
eye disease, the following routes are most commonly considered:
►intravitreal injection (Figure 1A)
►subretinal injection (Figure 1B)
►subtenon's injection
►systemic delivery
All of these have been considered but the most
likely route of delivery in initial trials (eg, the forthcoming gene therapy
trial for Leber's Amaurosis) will be subretinal or intravitreal.3,14
However, the AdPEDF.11 trial has been expanded in animal models (pig) to assess
the efficacy of the subtenon's route of delivery with encouraging results;15
if successful, this route would allow for a relatively non-invasive method for
therapeutic delivery.
Issues that are relevant to the delivery
technique include tissue penetration of the vector, the ability of the vector to
enter the target cell by endocytosis or other means, the resistance of the gene
and the agent to intracellular hydrolases to survive, and the half-life of the
agent in the target tissue. In principle, genes that are incorporated in the
host genome should be able to maintain expression for the life of the host cell,
although this is not always the case.
POTENTIAL GENE
TARGETS FOR AMD
As described above, the initial steps in the
development of AMD are not understood completely, thus making it difficult to
outline a logical gene therapy approach to the management of early disease.
However, previous studies have suggested a link between mutations in genes for
complement factor H, factor B, hemicentin, fibulin, and/or ABCA4 that may confer
or modify a patient's risk of acquiring the disease. Clinically, disease is
associated with lipofuscin accumulation, lipid accumulation, and thickening and
collagen cross-linking within Bruch's membrane; visual loss occurs through the
development of geographic atrophy or choroidal neovascularization. Currently,
the limited gene therapy trials in AMD are targeted at controlling
neovascularization in advanced exudative AMD. Future studies may be directed at
geographic atrophy.
■Gene Therapy for Neovascular AMD.
The growth of abnormal choroidal vessels that characterize the end-stage
of AMD is an obvious target for the application of therapies, specifically
towards the VEGF molecule. To date, there has been just 1 gene therapy trial
published in relation to AMD. Campochiaro and colleagues reported earlier this
year on the results of their phase 1 clinical trial using adenoviral
vector-delivered pigment epithelium-derived factor (AdPEDF.11) via intravitreal
injection.3 This dose-escalation study was specifically designed to
assess safety; a therapeutic benefit (less progression in growth of lesion size)
was detected and doses >108 PU were used. No serious local or
systemic adverse events were encountered. Mild inflammation and moderate
pressure rises were reported, with one patient developing marked (grade 3)
inflammation. Serum antibodies to adenovirus were unchanged in 27 patients, with
only 1 patient demonstrating a temporary titre rise that returned to normal. It
must be noted that only 28 patients (3 patients per dose level except 109.5
PU where 7 patients were enrolled) were treated in this study and safety data
with trend effect are reported. Further studies with a larger cohort that look
specifically at efficacy are ongoing.
This study coupled with the recent paper on gene
therapy for retinoblastoma14 establishes adenovirus as a good
potential vector for delivery of ocular gene therapy.
Other targets for treatment and/or prevention of
neovascular AMD are listed in Table 2. Adeno-associated viral (AAV)
delivery of angiostatin16 a plasminogen-derived fragment that is a
potent inhibitor of angiogenesis17 or Flt-14 has been
shown to suppress CNV in animal models. Other gene therapy based agents include
the siRNA approach to inhibiting VEGF18 and recruitment is currently
ongoing for the Cand5 siRNA (inhibitor of VEGF mRNA: Acuity Pharmaceuticals)
trial.
■Gene Therapy for Geographic Atrophy.
In AMD, changes lead to the accumulation of intracellular (RPE)
lipofuscin and extra cellular deposits (drusen and basal laminar deposits) that
contain complement complexes and other inflammatory markers19. These
lead to alterations in the nature and thickness of Bruch's membrane and
oxidative stress,20 leading to cellular changes in the RPE, inducing
apoptosis with secondary neural retinal degeneration. An approach in geographic
atrophy may be to introduce therapeutic genes into the host RPE to enhance their
survival in this environment. This may be achieved in this environment using
virally delivered anti-apoptotic factors eg, super-oxide dismutase 2 (SOD2);
caspase inhibitors or agents that will increase resistance to apoptosis such as
integrins (eg, av�3).
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Figure 3. Gene regulation using a
physiologic promoter.
Soluble VEGF
receptor Flt-1 is downstream on the gene and expressed
at normal levels during normoxia. In hypoxia, the HRE is
upregulated (switched on), increasing the level of local
Flt-1, which binds with VEGF and reduces the local VEGF-mediated
effects.
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CAN WE PREVENT AMD WITH GENE THERAPY?
It is apparent that diet and environmental
factors such as smoking play a role in the course of the disease, but there is
now strong evidence for a hereditable component.21-23 Recently,
definitive gene mutations have been identified as common to a large number of
AMD sufferers: complement factor-H,24,25 with modifiers (Factor B),26
HEMICENTIN-1,(27) fibulin, and ABCA4. These are exciting developments and may
lead to a greater understanding of the underlying pathogenic mechanisms that are
important in the development of the disease. Several authors have proposed a
role for abnormal complement activation in the development of AMD; this role is
supported by the presence of complement components in the extracellular milieu
in AMD eyes;19 the development of AMD-like deposits in a knockout
mouse28 and the presence of CD46, an important regulator of
complement activation that is associated with the cell attachment protein �1�integrin,
on the basal RPE surface.29 We are now in a better position than ever
to appreciate the exact pathogenesis of AMD; this should hopefully lead to
better therapies. In principle, it may be possible to alter specific protein
levels in the macula by performing gene therapy in patients with a definitive
AMD associated gene mutation; such an application may be possible in the future.
It is interesting to note that it may be possible
to target intraocular lipofuscin accumulation with gene therapy in the future.
There is a growing amount of evidence for the existence of microbes that can
metabolize intracellular lipofuscin.30 Based on these observations,
de Grey proposed a therapeutic approach to the age-related storage diseases,
involving the identification of enzymes capable of degrading lipofuscin. These
may be introduced into human cells by gene therapy as a means of removing the
offending material.31 This future strategy is similar to the strategy
currently employed for FDA-approved trials of enzyme-replacement therapy for 2
major lysosomal storage disorders, non-neuronopathic Gaucher disease and Fabry
disease. Whether this approach will prove to be helpful in AMD remains to be
determined.
SUMMARY
Research into AMD has advanced greatly in the
past decade, particularly with the application of molecular techniques. The
recent completion of the Human Genome Project will facilitate identification of
gene mutations that may predispose patients to AMD, and in the future it may be
possible to use gene therapy to modify the expression of these genes. The most
immediate application of gene therapy in AMD will involve targeted therapy aimed
at controlling late manifestations of this disease, such as geographic atrophy
and choroidal neovascularization. The potential to deliver gene therapy to the
eye without systemic side effects is great. We are currently enjoying the
benefits of anti-VEGF agents; the use of gene therapy to modulate early and
advanced disease is the next horizon in the management of this destructive
condition.
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Figure 4. Tet-Off and Tet-On gene
switch system.
A. Tet-Off system. In the absence of a tetracycline (eg,
doxycycline), the tTA binds to the TRE and activates
transcription of the gene of interest, eg, PEDF.
Exogenous doxycycline inactivates tTA and gene
transcription is silenced.
B. Tet-On system.
In this system, the rtTA is maintained in an inactive
state in the absence of doxycycline and transcription of
the gene is silenced. In the presence of drug, the rtTA
binds to the TRE and activates the transcription of PEDF.
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David Keegan, MD, PhD, is in the
Department of Ophthalmology, Columbia University, New York, NY. He can be
e-mailed at djk2112@columbia.edu. Lucian V. Del Priore, MD, PhD, is a professor
and Robert L. Burch III Scholar in the Department of Ophthalmology, Columbia
University, New York, NY. Dr. Del Priore is also in private practice at
Vitreous-Retina-Macula Consultants of New York in Manhattan. He can be e-mailed
at lvdelpriore@gmail.com. Neither
author has a financial interest in any of the information contained in this
article.
Retinal Physician, Issue: May 2006