REVIEW ARTICLE
Gene Therapy for Age-Related Macular Degeneration
Ian Jeffery Constable, FRANZCO,* Mark Scott Blumenkranz, MD, Steven D. Schwartz, MD,
Sam Barone, MD, Chooi-May Lai, PhD, and Elizabeth Piroska Rakoczy, PhD
Abstract: The purpose of this article was to evaluate safety and signals of
efficacy of gene therapy with subretinal rAAV.sFlt-1 for wet age-related
macular degeneration (wet AMD). A phase 1 dose-escalating single-
center controlled unmasked human clinical trial was followed up by exten-
sion of the protocol to a phase 2A single-center trial. rAAV.sFlt-1 vector
was used to deliver a naturally occurring antivascular endothelial growth
factor agent, sFlt-1, into the subretinal space. In phase 1, step 1 randomized
3 subjects to low-dose rAAV.sFlt-1 (1  1010 vector genomes) and 1 sub-
ject to the control arm; step 2 randomized an additional 3 subjects to treat-
ment with high-dose rAAV.sFlt-1 (1  1011 vector genomes) and 1 subject
to the control arm. Follow-up studies demonstrated that rAAV.sFlt-1 was
well tolerated with a favorable safety profile in these elderly subjects with
wet AMD. Subretinal injection was highly reproducible, and no drug-
related adverse events were reported. Procedure-related adverse events
were mild and self-resolving. Two phakic patients developed cataract and
underwent cataract surgery. Four of the 6 patients responded better than
the small control group in this study and historical controls in terms of
maintaining vision and a relatively dry retina with zero ranibizumab
retreatments per annum. Two patients required 1 ranibizumab injection
over the 52-week follow-up period. rAAV.sFlt-1 gene therapy may prove
to be a potential adjunct or alternative to conventional intravitreal injection
for patients with wet AMD by providing extended delivery of a naturally
occurring antiangiogenic protein.
Key Words: macular degeneration, gene therapy, adeno-associated virus, sFlt
(Asia Pac J Ophthalmol 2016;5: 300303)
T he therapeutic potential for gene therapy has attracted extensive
research and clinical trials in otherwise untreatable rare retinal
diseases, including Leber congenital amaurosis, choroideremia,
and X-linked retinoschisis.18 Conventional gene therapy aims
to replace an absent or defective gene, but the potential also exists
to upregulate naturally occurring bioactive molecules or insert
DNA to manufacture new ones. This modified gene therapy opens
up the potential to treat common diseases such as age-related mac-
ular degeneration (AMD).
Age-related macular degeneration is the leading cause of
blindness in the elderly population in the Western world, and vas-
cular endothelial growth factor (VEGF) has been identified as one
of the critical activators of neovascularization in wet AMD. The
suppression of subretinal neovascular leakage by monthly intravit-
real injection of anti-VEGF agents in wet AMD has proven to be
one of the major advances in ophthalmology in recent times. How-
ever, monthly bolus therapy with anti-VEGF agents causes peaks
From the *Lions Eye Institute; Sir Charles Gairdner Hospital, Nedlands; Cen-
tre for Ophthalmology and Visual Science, The University of Western
Australia, Crawley, Western Australia, Australia; University of California
Los Angeles, Los Angeles; Byers Eye Institute at Stanford, Palo Alto;
and Avalanche Biotechnologies, Inc, Menlo Park, CA.
Received for publication March 28, 2016; accepted May 29, 2016.
Supported by the National Health and Medical Research Council of Australia
and the Avalanche Biotechnologies, Inc, California.
The authors have no conflicts of interest to declare.
Reprints: Ian Jeffery Constable, FRANZCO, Lions Eye Institute, 2 Verdun St,
Nedlands, Western Australia, 6009 Australia. E-mail: ian.constable@lei.org.au.
Copyright 2016 by Asia Pacific Academy of Ophthalmology
ISSN: 2162-0989
DOI: 10.1097/APO.0000000000000222
300 www.apjo.org Asia-Pacific Journal of Ophthalmology Volume 5, Number 4, July/August 2016
Copyright 2016 Asia Pacific Academy of Ophthalmology. Unauthorized reproduction of this article is prohibited.
and troughs of drug and consequent cyclical effects on vision and
center point thickness (CPT) of the macula. This effect corre-
sponds to the observation in randomized dosing trials, which com-
pared prescribed monthly injections with as-needed injections,
that more frequent injections are associated with better out-
comes.9,10 Therefore, continuous anti-VEGF suppression may
be superior to intermittent treatment if feasible and safe. Further-
more, the burden of monthly visits and injections on the elderly
population and their caregivers is such that insufficient compli-
ance and undertreatment are very common.1013 Current treatment
regimes, which in many cases continue injections indefinitely, also
result in an enormous and growing cost burden.14,15 Therefore, an
alternative therapy that could last much longer is clearly needed.
One long-term strategy for anti-VEGF delivery is through
modified gene therapy. By inserting an anti-VEGF complemen-
tary DNA construct encoded in a viral vector into photorecep-
tors and retinal pigment epithelial cells adjacent to a choroidal
neovascular membrane, it is possible to upregulate the VEGF in-
hibitor production locally. A variety of antiangiogenic molecules
have been explored for this purpose, including bevacizumab and
variants of soluble Flt-1 (sFlt-1). Soluble Flt-1 is 1 of the 2 known
endogenously expressed and secreted VEGF inhibitors, and it
binds and neutralizes VEGF-A2 with very high affinity (10 pM).16
Soluble Flt-1 inhibits VEGF by sequestering VEGFs angiogenic
action and by heterodimerizing with the Flt-1 and Flk-1/KDR ex-
tracellular ligand-binding region,17 thereby blocking the phos-
phorylation and activation of downstream signal transduction
pathways for endothelial cell proliferation. Aflibercept (Eylea),
used in treating wet AMD, is a synthetic fusion construct that
binds the same receptor complex as sFlt-1.
BACKGROUND
To test the hypothesis that enhanced endogenous production
of sFlt-1 in the eye may effectively block VEGF-mediated ef-
fects on the retina, we designed and constructed a recombinant,
replicative-deficient adeno-associated viral (AAV) vector of sero-
type 2 encoding a truncated sFlt-1 (rAAV.sFlt-1).18
Subretinal delivery of rAAV.sFlt-1 reversed pathology in
induced neovascularization in a transgenic mouse model of
VEGF-driven retinal neovascularization (trVEGF029).18,19 In-
travitreal administration, a less invasive route of delivery to the
retina, has been investigated. However, current intravitreal AAV2
virus constructs for gene therapy principally transduce the adja-
cent retinal ganglion cells and do not readily penetrate the retina
to the site of pathology found in most retinal conditions. After in-
travitreal injection, only a small ring of ganglion cells around the
central retinal area are transduced,20,21 whereas after subretinal in-
jection, the retinal photoreceptor cells and retinal pigment epithe-
lial cells in the area elevated by the fluid bleb were transduced, as
evidenced by subsequent expression of a reporter protein.22
Therefore, most current clinical gene therapies under evaluation
for retinal conditions utilize subretinal injection.23 Only 3 AAV-
mediated phase 1 trials currently listed on clinical trials.gov are
evaluating intravitreal injection. Two are for the expression of
retinoschisin in patients with X-linked congenital retinoschisis
and 1 for Leber hereditary optic neuropathy to overcome the
Asia-Pacific Journal of Ophthalmology Volume 5, Number 4, July/August 2016
FIGURE 1. rAAV.sFlt-1 phase 1 and 2A trial design.
ND4 mutation in mitochondrial DNA. These are special situations
where the internal limiting membrane may be disrupted or defi-
cient, allowing access from the vitreous.
In primate experiments carried out in Singapore, we found
that a single subretinal injection of rAAV.sFlt-1 led to prolonged
expression of sFlt-1 for 17 months or more.19 When injected un-
der the retina, rAAV.sFlt-1 inhibited laser-induced submacular
neovascularization, and the effect lasted at least 18 months.24
There was no clinical or laboratory evidence of toxicity after
subretinal injection of rAAV.sFlt-1 in the primates.25
MATERIALS AND METHODS
We next designed the first ocular gene therapy human clini-
cal trial in Australia, which was approved by the Therapeutic
Goods Administration in 2011 (NCT01494805). The trial was
performed at Lions Eye Institute in Nedlands, Australia. Ap-
proval was obtained from the University of Western Australia
Institutional Biosafety Committee and the Sir Charles Gairdner
Hospital Human Ethics Committee. The tenets of the Declara-
tion of Helsinki were observed, and all subjects provided writ-
ten informed consent.
The phase 1 study was a single-center, randomized clinical
trial to investigate the safety and potential activity of 2 different
dose levels of rAAV.sFlt-1 in subjects with wet AMD.26 Step 1
randomized 3 subjects to low-dose (LD) rAAV.sFlt-1 (1  1010
vector genomes or vg) and 1 subject to the control arm. The
subretinal gene therapy delivery procedure has been described.26
After allowing 8 weeks to observe any adverse effects from the
LD, step 2 randomized an additional 3 subjects to treatment with
high-dose (HD) rAAV.sFlt-1 (1  1011 vg) and 1 subject to the
control arm. Randomization was accomplished by sequential
study group assignment according to a randomization list gener-
ated before the study and held off-site. Subjects and procedure
staff were not masked to treatment received. Staff performing
the assessments were masked to the 2 groups of subjects at
study visits.
Eligible patients were aged 65 years or older, of either sex,
and had been diagnosed with wet AMD meeting the inclusion
criteria. Eligibility criteria included AMD secondary to active
subfoveal choroidal neovascularization as evidenced by leakage
on fluorescein angiography (FA) and fluid on optical coherence
tomography (OCT), with advanced disease as reflected by best-
corrected visual acuity (BCVA) of 3/60 to 6/24 in the study eye.
Patients were anti-VEGF therapy treatment experienced, and
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Gene Therapy for Age-Related Macular Degeneration
there was no washout period for anti-VEGF therapy before
the baseline visit.
All subjects received 0.5 mg ranibizumab (RBZ) by intravit-
real injection at baseline (day 0) and week 4 (Fig. 1). On study day
7 (week 1 visit), subjects randomized to HD or LD underwent
core vitrectomy and posterior vitreous detachment and received a
100-L subretinal injection of the appropriate dose of rAAV.sFlt-1
(Fig. 2). A 41-gauge needle connected to extension tubing with a
combined dead space of 250 L (DORC, the Netherlands) was used
with a 1-mL syringe to deliver the subretinal dose, propelled by a
short column of air. An 8-week observation period was enforced be-
tween LD and HD treatments to assess dose-dependent safety. This
period provided an adequate window to allow for the start of sFlt-1
protein expression and for any potential early immune response to
develop. Beginning at week 8, subsequent study visits occurred ev-
ery 4 weeks through week 52 (total of 12 visits). Adverse event in-
formation was solicited from subjects at each visit and reported to
the principal investigator (I.J.C.) who determined whether they were
related to gene therapy, RBZ therapy, study procedures, or
were unrelated.
During the study period, subjects were permitted retreatment
with RBZ according to prespecified criteria based on BCVA,
OCT, and FA.
After the 52-week primary end point, long-term follow-up
through 36 months was continued with protocol-specified visits
at 18 and 36 months. During this follow-up period, intravitreal
RBZ was administered at or between study visits according to
RBZ retreatment criteria.
The human trials were then extended to a phase 2A study
using the same rAAV.sFlt-1 construct and study design. It in-
volved 32 subjects with active wet AMD randomized to either
rAAV.sFlt-1, 1  1011 vg (n = 21), or the control group
(n = 11). Eligibility criteria in the phase 2A study were expanded
reflecting the evidence of safety garnered from the phase 1 study.
Subjects included were 55 years or older, with a BCVA worse than
6/9 secondary to wet AMD due to active subfoveal choroidal neo-
vascularization demonstrated by leakage on FA or the presence of
fluid on OCT. Most subjects had previously received anti-VEGF
therapy, and a washout period for the anti-VEGF was not required
before the baseline visit.
All 32 subjects received intravitreal RBZ 0.5 mg in the study
eye at baseline (day 0) and at the week 4 visit; the subjects in the
gene therapy group received 100-L subretinal injection of
rAAV.sFlt-1 after a core vitrectomy at day 7. All subjects were
assessed every 4 weeks out to the 52-week primary end point;
study assessments included ophthalmic examination, BCVA, and
OCTevery 4 weeks. Fluorescein angiography was performed quar-
terly. Subjects were permitted retreatment with intravitreal RBZ
FIGURE 2. Subretinal injection of rAAV.sFlt-1 in a patient with
advanced wet AMD.
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Constable et al Asia-Pacific Journal of Ophthalmology Volume 5, Number 4, July/August 2016
according to the prespecified criteria. The long-term follow-up is
planned out to 36 months.
Safety Laboratory Tests and Assessment of
rAAV.sFlt-1 Biodistribution and Immune
Responses to AAV2
Laboratory tests included routine hematology; renal and he-
patic function tests; urine protein; serum IgM, IgG, and IgA levels
and protein electrophoresis; and enumeration of peripheral blood
lymphocyte subsets (B cells: CD19+; T cells: CD3+, CD4+, CD8+;
and natural killer cells: CD3, CD16+, CD56+). Biodistribution
of rAAV.sFlt-1 was assessed by quantitative polymerase chain re-
action for rAAV.sFlt-1 DNA, AAV2 capsid detection by ELISA,
and sFlt-1 levels in tears from treated and fellow eye, serum,
urine, saliva, and vitreous samples where available by ELISA.
Adeno-associated viral vector 2specific immune responses assessed
included neutralizing antibodies (nAb), IgG antibodies to capsid
proteins by ELISA, and T-cell responses to 3 pools of peptides
from capsid proteins by interferon- ELISpot assay.
These studies are primarily designed to assess the safety and
tolerability of rAAV.sFlt-1 in subjects with wet AMD. Secondary
objectives are to assess the effect of rAAV.sFlt-1 using the explor-
atory end points of BCVA and number of RBZ retreatments re-
quired. Other prespecified end points included retinal CPT and
central subfield thickness on OCT. Biodistribution of rAAV.sFlt-1
and the immune response to AAV2 are also assessed.
RESULTS
Subretinal injection was successfully achieved in all eyes.
The placement of the 41-gauge needle under the retina was
assisted by high magnification achieved by a flat contact lens,
which gave superior depth perception compared with the wide-
angle indirect viewing systems. Damage to the underlying retinal
pigment epithelium caused a minor subretinal bleed in a minority
of eyes, but this did not affect vision.
The size and shape of the subretinal bleb varied in height and
circumference. Only 1 eye had extension of the subretinal bleb un-
der the central foveal area.
The phase 1 study passed all primary safety end point
criteria, and the biologic signals measured were suggestive of bio-
logic activity. No cardiovascular or other systemic adverse events
were recorded. Adverse events were related to the study procedure
and included subconjunctival hemorrhage, subretinal hemorrhage,
FIGURE 3. A, Visual acuity (number of letters on x-axis) after subretinal injection of rAAV.sFlt-1 in wet AMD over 36 months in a patient in the
LD group. Three RBZ rescue injections were required over 3 years. B, Same patient showing intraretinal fluid on OCT at entry, which was dry
36 months after gene therapy.
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and mild cell debris in the vitreous. Two phakic patients required
cataract surgery at weeks 36 and 44, respectively.
Four of the 6 gene therapy subjects required no subsequent
RBZ retreatments during the first year; 2 subjects required 1
RBZ retreatment each. Center point thickness improved early
and was maintained through month 12 in all gene therapy sub-
jects. Five of the 6 subjects gained vision at 1 year. The 1 subject
whose vision did not improve was noted to have clinically signif-
icant subretinal fibrosis at baseline. Four of the 6 patients remain-
ing in the trial at 3 years had on average less than 2 RBZ
retreatments per year, with a mean BCVA change from baseline
to month 36 of +0.5 letters (Fig. 3).
The phase 2A study was designed to evaluate safety and tol-
erability with the higher dose rAAV.sFlt-1 in a larger, more repre-
sentative wet AMD patient population.
This was an aged population with an average of 10 to 11
prior injections, persistent wet AMD, and established sequelae
such as subretinal fibrosis and retinal pigment epithelial detach-
ment. Phase 2A subjects had better vision and lower CPT values
at baseline overall. Three eyes with no prior anti-VEGF injections
were also included. Detailed data analysis of the phase 2A study at
the 1-year follow-up point is currently being assessed and not
yet available.
DISCUSSION
Subretinal injection of AAV gene product was found to be
well tolerated in an aged population with persistent wet AMD.
The procedure, which included a core vitrectomy, was carried
out under local anesthetic and required only 3 days of postopera-
tive antibiotic and steroid drops. Subretinal hemorrhage occurred
in a minority but cleared without any loss of vision. Cataract de-
veloped in phakic eyes and was removed by 10 months in all cases
with no deleterious effects on vision or the control of wet AMD.
Extensive monthly laboratory screening revealed no toxicity or
deleterious immune responses.
These gene therapy trials are the first in elderly wet AMD pa-
tients to demonstrate safety and signals of biologic activity. Ethics
dictate that patients with advanced wet AMD be enrolled in these
pioneering trials where the primary end point is safety. Five of the
6 gene therapytreated subjects showed improvement in vision
despite previously responding incompletely to a mean of 10 anti-
VEGF injections. Considering the long-term anti-VEGF treatment
history of the participating subjects before entering the trial, it
seems that the sustained delivery of sFlt-1 might improve patient
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Asia-Pacific Journal of Ophthalmology Volume 5, Number 4, July/August 2016
response and perhaps could result in better visual outcomes among
poor responders. With a robust safety proven in the phase 1 trial, fu-
ture gene therapy trials might select treatment-naive patients or
choose those who clearly respond to an anti-VEGF injection before
entering the trial.
In addition, further work in the laboratory and the clinic is
being carried out to help better define the key variables that are
critical to achieving more favorable outcomes. These include the
dose, volume, and location of the subretinal bleb, which can all in-
fluence the extent of upregulation of sFlt-1. Variations in delivery
technique, such as choice of tubing, cannula, needle, and automa-
tion for injection, may all better standardize the subretinal surgery.
rAAV.sFlt-1 gene therapy may prove to be a potential adjunct
or alternative to conventional intravitreal injection for patients
with wet AMD by providing extended delivery of sFlt-1.2729
One significant advantage of rAAV.sFlt-1 treatment is that it de-
livers a naturally occurring anti-VEGF substance, sFlt-1, that is
capable of addressing the imbalance between VEGF and sFlt-1
in the eye. Other alternative gene therapy approaches, including
novel AAV subtypes that allow intravitreal delivery and gene
editing for classic gene therapy, may also play a role in future.
ACKNOWLEDGMENTS
The authors sincerely thank Ms. Cora Pierce, RN, Lions Eye
Institute Perth, for her skilled management of the patients and also
Dr. Vignesh Raja and Dr. Sendhil K. Somasundaram, Sir Charles
Gairdner Hospital, Nedlands, Australia, for participating in the
surgery and some of the follow-up studies.
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