41
Niche, 2013; 2: 41-7 • DOI: 10.5152/niche.2014.188
Update on Gene Therapy in
Ophthalmology
Gökçen Gökçe1, Güngör Sobacı2
Review
Abstract
Gene therapy, in its current form, seems to be a promising treatment option for almost all ocular diseases, because the eye
represents a unique target organ for gene therapy due to both structural and functional properties. Encouraging results
obtained for Leber’s congenital amaurosis, a hereditary retinal disease in childhood that is otherwise destined to blindness,
focused not only ophthalmologist’s but also other clinician’s interest in gene therapy. In this review, we will provide an
overview of current gene therapy trials in the trials in ophthalmology.
Key words: Gene therapy, inherited disease, cornea, retina
Introduction
After the first human gene therapy trial by Anderson in 1990, the number of gene therapy trials has
increased incredibly, from 12 in March 1992 to 1024
protocols and 5000 patients in March 2005 and
3723 protocols with 1134 studies in phase 3 in October 2014. The potential of gene therapy in ophthalmology has come to the fore, particularly in the past
decade, and extensive improvements have been
made in this field. Suicide gene therapy for retinoblastoma by Hurwitz in 1999 was the first gene
therapy for the eye. The official website of clinical
trials (www.clinicaltrails.gov) reveals 122 ongoing
gene therapy studies, most of which are pertinent
to retinal disease of hereditary origin. On the other
hand, only RPE-65 gene therapy for Leber’s congenital amaurosis (LCA) reached phase 3. Current
clinical gene therapy trials and related companies/
institutions are tabulated elsewhere (1).
Department of Ophthalmology, Kayseri
Military Hospital, Kayseri, Turkey
1
Department of Ophthalmology, Hacettepe
University Faculty of Medicine, Ankara,
Turkey
2
Submitted: 30.12.2014
Accepted: 09.01.2015
Correspondence: Dr. Güngör Sobacı,
Department of Ophthalmology,
Hacettepe University Faculty of Medicine,
Ankara, Turkey
Phone: +90 90 530 527 61 77
E-mail: gsobaci@gmail.com
©Copyright 2013 by Cellular Therapy and
Regenerative Medicine Society
Available online at www.nichejournal.org
Gene therapy requires a viable and responsive target, and it is not confined only to the genetic disease. Any disease that could benefit from the local
production of a genetically engineered protein,
peptide, RNA, or RNA fragment would be a potential candidate for gene therapy. Strategies for human ocular gene therapy include the following:
(i)Gene replacement used for the autosomal
recessive (AR) diseases of LCA (RPE-65), Stargardt’s disease (ABCA4), and Usher syndrome
(USH) type 1B (MYO7A)
(ii)Gene inactivation/edition in structural or autosomal dominant (AD) disease.
Enhancement of endogenous DNA (protein) in
age-related macular degeneration (ARMD) and
retinitis pigmentosa (RP) by genetically-modified
drug delivery systems and delivery of engineered
transgene VEGF receptor (sFlt-1)-IgG1-Fc portio,
which has a well-characterized binding ability to
VEGF and successful use in ARMD, have also been
mentioned in this context.
The advantages of the eye in gene therapy applications are as follows:
a.Visual functions can be measured directly
and noninvasively; therefore, the success of
treatment can be evaluated objectively,
b.Specific gene mutations responsible for
many genetic eye diseases are already
known,
c. The diseased tissues can easily be observed,
d.Because the eye is a relatively self-limiting
organ, local transfer methods can be applied
in gene transfer, and systemic toxicity can be
minimized,
e. The symmetrical and bilateral nature of the
eye facilitate the control of success in clinical
studies.
Structurally and functionally, the inner coating of
the eye, the retina, has several characteristics that
are advantageous for the practice and monitoring
of gene therapy. Gene therapy has potential applications for both acquired and hereditary diseases
of the retina. Among them, inherited retinopathies
have gained special interest mostly because they
have the outcome of blindness in early adult life,
resulting in socioeconomic burden in the public.
42
Gökçe and Sobacı. Gene Therapy in Ophthalmology
Inherited retinopathies (including all types) found in all layers of
the retina are relatively common (almost 1/2000) and currently
untreatable diseases. A considerable number of them are inherited as a monogenic disorder, which is preferable in gene therapy
aiming for a cure. In addition, the retina’s compatibility with gene
transfer has made transduction of different retinal cell layers via
viral and nonviral vectors possible.
Genetic retinal diseases are of a vast variety and have various inheritance patterns. In studies in the past decade, it was shown
that certain genetic retinal diseases were more convenient than
others in terms of the application of gene therapies. Specifically,
diseases that affect retinal pigment epithelium (RPE) and cause
slow-progressing photoreceptor loss are found to be more suitable for gene therapy (2). Gene transfer for photoreceptors is,
however, obstructed by the lack of phagocytosis function in photoreceptors, the presence of lipid intensive discs in the outer segment, and the location of nuclei in the inner segment.
Gene Therapy for Retinitis Pigmentosa
RP is a hereditary retinal disease that occurs in an average of
1/2,500-1/4,000 people; it is characterized by typical tapetoretinal degenerative changes (spindle or bone corpuscle pigmentation) in the retina that are associated with symptoms of
peripheral and central vision loss. Except some syndromic cases,
mutations that are heterogeneous in nature are found in all cases. The basic pathological mechanism that causes the death of
photoreceptors in RP is apoptosis, as is the case in all retinal dystrophies. Therefore, the Bcl-2 gene, one of the genes that control
apoptosis in mammalian tissues, has been of clinical interest in
gene therapy studies.
Many different animal models for RP are available in the literature.
The Rd mouse is the first defined AR mutant. The other model
called the rds mouse is AR, and there is loss in the functional gene
of peripherin 2. The RCS mouse is a frequently studied retinal degeneration model that has an RPE defect. Although RPE cells are
defective, the first dying cells are photoreceptors.
The inheritance of RP is AD in 30-40% of patients and AR in 5060% of patients, whereas there is X chromosome inheritance in
5-15% of patients. In 50% of the AD cases and 30% of the AR cases, the responsible gene has yet to be revealed. X-linked RP is the
severest form of the disease and usually involves the RPGR gene
mutation (3). In a recent animal study, successful results were reported for X-linked RP through RPGR gene therapy (4).
The MERTK gene mutation is usually observed in AR RP (5). Although short-term functional improvements were achieved in
experimental applications, the transferred MERTK gene did not
lead to a long-term and permanent effect due to the molecular
structure of the disease (6, 7).
The rhodopsin gene mutation is generally observed in AD RP and
shows a heterogeneous disease phenotype that varies from the
mild to severe form (8). A total of 20-30% of patients with AD RP
have the rhodopsin mutation. About 120 rhodopsin mutations
divided into 7 classes have been reported so far, the most common being the class 2 (P23H) mutation (9). Although gene silenc-
Niche, 2013; 2: 41-7
ing regarding rhodopsin is in the works, a long-lasting and permanent effect has yet to be obtained (3).
Gene Therapy for Leber’s Congenital Amaurosis
LCA is a heterogeneous group of AR diseases characterized by
early-onset vision disability, nystagmus, and progressive degeneration in hypermetropic eye with flat ERG and severe vision loss
in most cases. About 400 mutations in 14 genes were detected
regarding LCA. Lesitin retinol acetyltransferase (LRET) mutations
constitute 0.5% of LCA cases, whereas RPE-specific 65 kDa protein (RPE65) constitutes 7-16% of these cases. LCA related to the
RPE65 gene mutation is the most extensively studied hereditary
retinal disease as yet, but it still entails many molecular uncertainties that are yet to be solved (10). In experimental animal studies
in the past few years, the ERG change by functional correction of
RPE65 with the duration of 3 years had been reported (11-13).
There are several ongoing phase I, II, and III studies in the United
States and Europe for LCA2 using a variety of vectors and protocols to treat LCA2, and many believe that Food and Drug Administration (FDA) approval is possible. Although the initial clinical
results have been encouraging and have led to improvements in
visual function lasting several years, there is evidence that the underlying retinal degenerative process in LCA2 may not be slowed
by the delivery of RPE65. It is noteworthy that younger age was
associated with a better outcome. On the other hand, Artur Cideciyan and colleagues recently demonstrated continued photoreceptor degeneration after therapy at a rate consistent with
the natural history of LCA2 (14). These results affirm the need
for long-term clinical studies of these therapies and for ongoing
research aimed at slowing the kinetics of the underlying degeneration, which may be perpetuated by “downstream” processes
unaffected by delivery of the gene product (15). Several other inherited retinal degenerations are in early phase clinical trials using a number of different viral vectors, including choroideremia,
AR RP, Stargardt’s disease, and USH (type IB), with many others
in preclinical animal studies. Most of the clinical work involves
transduction with AAV or lentiviral vectors. Currently, long-term
LCA animal model data suggest ongoing production of RPE65,
but there is no regulation of the kinetics of gene production. For
LCA2, this may not matter, but for other disease states, retinal
function may be more sensitive to the “therapeutic index” of the
gene product.
Mutations in MERTK, which is a gene encoding tyrosine kinase
receptors, constitute 0.6% of the LCA cases. Due to a defect of this
gene that controls the phagocytosis function of the RPE, toxic debris is accumulated in the subretinal area due to substantial deterioration of the phagocytosis mechanism. In experimental animal
studies, a partial short-term recovery has been observed in the
retinal morphology through MERTK gene therapy (6, 16).
Mutations related to photoreceptors constitute 53% of the LCA
cases. The most frequent mutations are CRB1 (10%), GUCY2D
(RETGC-1) (12%), and CEP290 (15%). Because new mutations
may be formed during CRB1 gene therapy, which may cause
severe retinal morphological disorders, gene therapy involving
this gene has not yet been suggested. In an experimental study
with the GUCY2D (RETGC-1) gene, partial preservation of retinal
morphology has been achieved (17). CEP290 is not suitable for
Niche, 2013; 2: 41-7
gene therapy studies for the time being due to its large molecular
structure.
Gene Therapy for Achromatopsia
Achromatopsia (ACHM) is caused by the progressive loss of cone
photoreceptors, leading to color blindness and poor visual acuity.
ACHM or in other words rod monochromatism is inherited as AR
pattern and occurs in 1/30,000 people. The most common gene
mutations are CNGB3 (50%), CNGA3 (25%), GNAT2, and PDE6C.
In recent experimental animal studies, a significant ERG change
with the duration of 2 months was achieved through CNGA3
gene therapy (18, 19). On the other hand, in GNAT2 gene studies,
the effect lasted 9 months (20, 21). Additionally, the ERG change
achieved by GNAT2 gene therapy was found to be much higher
than that achieved by CNGA3 gene therapy. In studies conducted with CNGB3, which is the most common gene mutation, the
ERG change was again achieved, although at lower rates (22, 23).
Animal studies and human clinical trials have shown that gene
replacement therapy with adeno-associate virus (AAV) is a viable treatment option for this disease. The use of a cone-specific
promoter may improve targeting in this condition. The chimeric
(IRBPe/GNAT2) promoter is more efficient and specific than the
synthetic, synGNAT2/GNAT2 promoter in this regard (24).
Gene Therapy for Usher Syndrome
USH is a genetic disease that occurs in approximately 1/25,000
people and is characterized by sensorineural hearing loss and
pigmentary retinopathy, and it is divided into three main types
according to the severity of the clinical presentation (25). Because type 1B USH is the most common (39-55%) and the most
severely progressing form, it has become the most frequently
studied subtype for gene therapy (26, 27). The MYO7A gene mutation is responsible for type 1B USH. However, because the gene
that causes the disease is rather large, its transportation is quite
difficult. USH is the most common form of inherited deaf-blindness, with a prevalence of approximately 1/6,000. Three clinical
subtypes (USH1-USH3) are defined according to the severity of
the hearing impairment, the presence or absence of vestibular
dysfunction, and the age of onset of RP. USH1 is the most severe
subtype and exhibits congenital severe hearing loss and onset
of RP before puberty. Currently, only the amelioration of hearing
deficiency is implemented, but no treatment exists for the sensoneuronal degeneration in the eye. In our studies, we are focusing
on the evaluation of gene-based therapies to cure retinal degeneration in USH1C patients:
(i) Gene augmentation using recombinant adeno-associated virus,
(ii) Genome editing by homologous recombination mediated by zinc-finger nucleases, and
(iii) Read-through therapy using novel designer aminoglycosides and PTC124.
Latter compounds target in-frame nonsense mutations, which
account for approximately 20% of all USH cases. All analyzed
gene-based therapy strategies have led to the restoration of
USH protein expression. These adjustments may be sufficient
to reduce the progression of retinal degeneration, which would
greatly improve the life quality of USH patients (28).
Gökçe and Sobacı. Gene Therapy in Ophthalmology
Gene Therapy for Stargardt’s Disease
Stargardt’s disease, the most common form of juvenile-type macula degenerations and its common type called fundus flavimaculatus, occurs in an average of 1/10,000 people. The mutation in
the ABCA4 gene, which is a photoreceptor transport gene that
functions in the retinoid cycle, causes Stargardt’s disease. This
mutation causes toxic A2E protein accumulation in the retinal
pigment epithelium. In animal model studies, it has been shown
that ABCA4 gene therapy may decrease A2E protein accumulation (29, 30).
Gene Therapy for Choroideremia
Choroideremia is an X-linked genetic disease that occurs in
1/50,000 individuals and develops into progressive neural retina,
RPE, and choroidal degeneration. The CHM gene mutation is responsible for choroideremia, which is a monogenic disease. The
CHM gene encodes the REP1 protein that is responsible for intracellular transport. Although the choroid is a tissue that is difficult
to reach for gene transfer, and it is still debated whether the REP1
protein obtained will be functional, clinical gene therapy studies
on choroideremia patients are at the final stage (31). In an experimental animal study in this field, slowdown of the degeneration
was achieved with the REP1 gene therapy (32). In preclinical studies in in vitro and in vivo models, adeno-associated virus 8-mediated gene therapy for choroideremia was found to be successful (33).
Gene Therapy for Gyrate Atrophy
Gyrate atrophy is an inherited condition in which areas of the
retina-the inner lining of the wall of the eye—become thin. Over
several decades, this degeneration of the retina causes tunnel
vision, night blindness, and other vision problems. Gyrate atrophy is caused by a defect in the gene responsible for producing
an enzyme, ornithine aminotransferase (OAT), that breaks down
the amino acid ornithine. As a result, excessive ornithine buildup causes retinal thinning. Currently, this condition can only be
treated with amino acid tablets and a very low-protein diet with
limited fruits and vegetables and more than 2,000 calories a day
from carbohydrates and fats. Some patients cannot maintain this
diet, and they need another treatment option. One possible alternative is to replace the defective gene with one that functions
normally, and this has been performed by National Eye Institute
as a phase I study in the “Safety and Efficacy of Transduced Keratinocytes for Possible Treatment of Gyrate Atrophy;” however,
completed study results have not been published yet.
Gene Therapy for Retinoblastoma
A suicide gene therapy protocol was initiated for bilateral retinoblastoma with the title “Phase I Study of AdV/RSV-TK Followed by
Valganciclovir for Treatment of Patients with Retinoblastoma” in
June 1999, and this study is still underway.
Gene Therapy for Age-related Macular Degeneration
Another major area of emphasis in gene therapy is the treatment
of wet ARMD. Although anti-VEGF drugs are currently successfully in treating wet ARMD, the cost and time involved in monthly
or semimonthly injections can be troublesome. A one-time treatment-the promise of gene therapy-is attractive. Several companies are at the forefront of research on the use of gene therapy
for wet ARMD. These include Genzyme; Avalanche Biotechnolo-
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Gökçe and Sobacı. Gene Therapy in Ophthalmology
gies, working with the Lions Eye Institute of Perth, Australia; and
Oxford BioMedica, in partnership with Sanofi. In wet ARMD, VEGF
plays a critical role because the blockade of VEGF is sufficient
to suppress the development of choroidal neovascularization
(CNV). A variety of antiangiogenic proteins oppose the actions
of proangiogenic factors, such as VEGF. Gene transfer to augment
expression of these endogenous inhibitors or related engineered
proteins is a potential alternative to suppress CNV and avoid frequent intraocular injections. Considerable preclinical and emerging clinical data suggest that this approach may be feasible. Early
phase 1 results of the secreted extracellular domain of VEGF receptor-1, sFlt-1, which is a naturally occurring protein antagonist
of VEGF formed by alternative splicing of the pre-mRNA for the
full-length receptor, have been shown to be promising when delivered to the vitreous (Avalanche trial) and the subretinal area
(Genzyme trial) by AAV2 as gene therapy. It is remarkable that
pigment epithelium-derived growth factor (PEDGF) gene therapy
was discontinued after phase ½ due to toxicity. The other study,
Endostatin/Angiostatin Co-Transfection by Oxford Biomedica as
retinostad, that is in phase 1/2 dose escalation uses lentiviral vectors for delivery to the subretinal area to overcome efficacy and
safety problems. The results seem to be promising (34).
Gene Therapy for Leber’s Hereditary Optic Neuropathy
Leber’s hereditary optic neuropathy (LHON) is the most common
mitochondrial disorder affecting retinal ganglion cells (RGC),
with an estimated prevalence of 1 in 25,000. The most mutations found in about 95% of LHON patients are located in ND1
(G3460A), ND4 (G11778A), or ND6 (T14484C) genes, which are
the three genes encoding for subunits of the respiratory chain
complex I. These mutations induce a decrease in ATP synthesis
while increasing oxidative stress. As vision loss often occurs in
one eye before affecting the second eye in the following months,
a potential therapeutic window for interventions seems to be
exist. Visual acuity appears to be the most suitable primary end
point for the planned clinical trial (35). GS010, which is an AAVbased gene therapy product containing the human ND4 gene
(rAAV2/2_ND4) in GenSight’s experiment, is now being investigated in the phase 1 clinical study, which began in February 2014.
Gene Therapy for Corneal Diseases
In terms of immunity, the isolated structure of the cornea makes
it an ideal tissue for gene therapy. It is also rather advantageous
for the monitoring of gene therapy because the cornea has a clear
structure and the stage of vascularization can be visually evaluated.
Gene Therapy for Corneal Graft Rejection
Penetrating keratoplasty is the main surgical method for the
treatment of corneal diseases that are pharmacologically incurable. Although the cornea is immunogenically isolated, corneal
graft rejection is still the leading cause of failure. Studies claim
that corneal gene therapy prevents graft rejection through antiapoptotic, anti-angiogenic, and immunomodulating effects (36).
Particularly, it is shown that lymphangiogenesis plays an important role in graft rejection pathophysiology, and the graft lifespan can be significantly prolonged through lymphangiogenesis
inhibition by neuropilin 2 inhibition and Flt23k and VEGFR1 activation (37-39). In immunomodulation studies, IL-10, cytotoxic T
Niche, 2013; 2: 41-7
lymphocyte antigen protein (CTLA), indoleamine-2, 3-dioxygenase (IDO) are often used (38, 40). In particular, it is reported that
IDO increases the graft lifespan significantly by blocking active T
cells at the G1 stage (41). Other studies have shown that p35 and
BCL-XL gene therapies and programmed cell death binding protein 1 (PD-L1) therapy prevent apoptosis in graft corneal endothelial cells, thereby prolonging the graft lifespan (40, 42-44). In
addition, through gene therapy, the graft endothelial cells, which
are normally at the stable G1 phase, can be passed to the mitosis
stage of S phase, thereby increasing their number (45).
Gene Therapy for Corneal Scar Formation
Corneal scar healing plays a key role in providing post-traumatic
corneal clarity and maintaining visual acuity. However, corneal
scar healing, which usually results in fibrosis and disorganized
scar tissue, occurs under the control of many cytokine and growth
factors. Among them, particularly, TGF-β is the cytokine that
plays the main role in scar formation (46). Therefore, TGF-β has
become the primary goal in gene therapies that aim to prevent
scar formation. In these studies, it was reported that treatment
with “Decorin,” which is a very important protein in the maintenance of corneal clarity, decreases TGF-β levels and prevents scar
formation to a great extent (47, 48). Another protein effective in
TGF-β signal activity is “Smad,” the treatment with which also has
proven to decrease scar formation (49).
Gene Therapy for Corneal Neovascularization
In neovascularization (NV) that occurs under the control of many
cytokine and growth factors, VEGF plays a key role. In addition,
PAX6 and sflt1 gene defects have been proven to be effective in
spontaneous NV formation (50-52). In recent years, it has been
shown that in experimental NV models, the endostatin protein
inhibits NV formation (53). Kringle-5 subsegment of plasminogen, angiostatin, IL-18, Vasohibin-1, CD36, neostatin, and decorin
are other molecules that inhibit corneal NV inhibition and are researched in gene therapy studies (51, 54).
Gene Therapy for Corneal Alkaline Burn
Corneal alkaline burn is a chemical corneal trauma that progresses into scar formation, NV, and corneal ulceration and causes severe vision loss. It has been shown that in studies, the peroxisome
proliferator activated receptor (PPARγ) gene reduces scar formation in corneal alkali burns (55).
Gene Therapy for Glaucoma
Glaucoma is an optical neuropathy with a progressive course that
causes severe vision loss. The discovery of the genetic basis of
glaucoma led to gene therapy studies. The main aim in such therapy is to decrease intraocular pressure and at the same time provide permanent neuroprotection. The key target tissues in these
studies are trabecular meshwork, ciliary body, ciliary epithelium,
muller cells, and retinal ganglion cells.
Gene Therapy that Targets the Trabecular Meshwork
As the trabecular meshwork has actin cytoskeleton structure,
the rho kinase (ROCK) pathway, actin-tropomyosin system, and
calmodulin protein have become prominent as primary targets in
gene studies that target the trabecular meshwork (56, 57).
Niche, 2013; 2: 41-7
Gene Therapy that Targets the Ciliary Body
Increase in the ciliary muscle tonus manipulates the trabecular
meshwork structure and at the same time changes the shape
of the lens, thereby increasing the uveal scleral outflow and decreasing intraocular pressure. It has been shown that stromelysin,
which is a type of matrix metalloproteinase, affects the trabecular
meshwork and the ciliary body, thereby increasing uveal scleral
outflow (58).
Gene Therapy that Targets the Retinal Ganglion Cell
The prevention of RGC damage, which is the main reason underlying vision loss due to glaucoma, has become a key target
in gene therapy studies. In particular, intravitreally administered
anti-apoptotic genes is expected to prolong RGC lifespan. BCLXL protein, caspase inhibitors (XIAP, BIRC4), brain-derived neurotrophic factor (BDNF), tropomyosin kinase-B (Trk-B), ciliary
neurotrophic factor (CNTF), and glial cell derivedneurotrophic
factor (GDNF) used in experimental glaucoma studies have been
reported to have various durations of neurotrophic effects on
RGCs (59-62).
Conclusion and Future Directions
Gene therapy has recently become a popular and promising
mode of treatment for genetically inherited eye diseases. However, despite all in vivo and ex vivo experimental animal studies,
a study that enables routine clinical use has yet to be provided.
The major restrictions of gene therapy are the short-term effect
of a given gene, the immune response of the recipient, toxicity of
the product, technical difficulties in preparation of the gene to be
administered in accordance with the target, considerable costs of
the therapy, and ethical problems. On the other hand, potential
problems associated with insertional mutageneity and tumorigenesis, a new homeostasis produced by gene therapy, and functionality of the gene and gene product(s) have to be considered
seriously for this life-long treatment modality. There are prospects
for commercially available gene therapies for retinal disease in the
near future, but much work remains to be performed to elucidate
all the molecular mechanism(s) in these genetically heteregenous
diseases. In this regard, monogenic retinal disease(s) seems to be
the best suitable target for gene therapy for the eye: the next step
will be to expand the indications available for treatment, and initiate treatment earlier in the disease process, which will require
excellent safety and efficacy data.
Peer-review: Externally peer-reviewed.
Acknowledgements: The authors would like to thank Ayşe Ünal
Ersönmez for professional language editing.
Author contributions: Concept - G.S.; Design - G.S., G.G.; Supervision - G.S.; Resource - G.S., G.G.; Data Collection&/or Processing - G.S., G.G.; Analysis&/or Interpretation - G.S., G.G.; Literature
Search - G.S., G.G.; Writing - G.S., G.G.; Critical Reviews - G.S., G.G.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study has
received no financial support.
Gökçe and Sobacı. Gene Therapy in Ophthalmology
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