Perspective

Ocular Gene Therapy with Adeno-associated Virus
Vectors: Current Outlook for Patients and Researchers

Geoffrey A. Casey1, BS (EE), BS (MolBiol); Kimberly M. Papp2, BS; Ian M. MacDonald1,3, MDCM

1Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Canada
2Faculty of Science, University of Alberta, Canada

3Department of Ophthalmology, Faculty of Medicine and Dentistry, University of Alberta, Canada

ORCID:
Geoffrey A. Casey: https://orcid.org/0000-0001-5012-7147
Ian M. MacDonald: https://orcid.org/0000-0001-7472-8385

Abstract
In this “Perspective”, we discuss ocular gene therapy – the patient’s perspective,
the various strategies of gene replacement and gene editing, the place of adeno-
associated virus vectors, routes of delivery to the eye and the remaining question - “why
does immunity continue to limit efficacy?” Through the coordinated efforts of patients,
researchers, granting agencies and industry, and after many years of pre-clinical studies,
biochemical, cellular, and animal models, we are seeing clinical trials emerge for many
previously untreatable heritable ocular disorders. The pathway to therapies has been
led by the successful treatment of the RPE65 form of Leber congenital amaurosis with
LUXTURNATM. In some cases, immune reactions to the vectors continue to occur, limiting
efficacy. The underlying mechanisms of inflammation require further study, and new
vectors need to be designed that limit the triggers of immunity. Researchers studying
ocular gene therapies and clinicians enrolling patients in clinical trials must recognize
the current limitations of these therapies to properly manage expectations and avoid
disappointment, but we believe that gene therapies are well on their way to successful,
widespread utilization to treat heritable ocular disorders.

Keywords:

J Ophthalmic Vis Res 2020; 15 (3): 396–399

INTRODUCTION

Patients with hereditary retinal disorders are
keenly interested in gene therapy. Many recent
media presentations feature patients who have
experienced apparent benefit from gene therapy

Correspondence to:

Ian M. MacDonald, MDCM, Department of
Ophthalmology and Visual Sciences, University of
Alberta, 7-030 Katz Bldg. Edmonton, Alberta T6G2E1,
Canada.
Email: macdonal@ualberta.ca
Received: Accepted:

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DOI: 10.18502/jovr.v15i3.7457

treatments, increasing public awareness of
gene therapy. It is true that gene therapy may
conceptually be an ideal method to treat heritable
eye diseases before irreparable damage has
occurred to the eye, but it is crucially important
to manage patient perspectives around their
participation in clinical trials. The leading
successful example of ocular gene therapy
is LUXTURNATM (voretigene neparvovec-rzyl),
an adeno-associated virus (AAV) vector treatment

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How to cite this article: Casey SA, Papp KM, MacDonald IM. Ocular Gene
Therapy with Adeno-associated Virus Vectors: Current Outlook for Patients
and Researchers. J Ophthalmic Vis Res 2020;15:396–399.

396 © 2020 JOURNAL OF OPHTHALMIC AND VISION RESEARCH | PUBLISHED BY PUBLISHED BY KNOWLEDGE E

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Ocular Gene Therapy; Casey et al

for Leber congenital amaurosis. LUXTURNATM
is now approved as a treatment in the USA
and the European Union. Unfortunately, many
patients believe that enrollment in a clinical trial
means “early access” to a treatment or cure,
when the trials have not yet reported fully their
outcomes. As an example, some patients do not
fully understand that LUXTURNATM is designed
to treat a single genetic disorder caused by
biallelic pathogenic variants in the RPE65 gene.
Patients with a progressive heritable eye disease
are a distinctly vulnerable population. They may
have incomplete knowledge of their condition and
partially formed opinions on risks and benefits of
experiments such as gene therapy. They carry
with them the hope that a trial may lead to
treatment but may also conflate the trial with finding
a cure. After the patient passes the screening
and consent process, they may face significant
personal challenges of a rigorous and frequent
follow-up schedule. They (or their families) may
not be completely prepared for the possibility of
harm or the systemic consequences of high-dose
immunosuppression. According to the partner of
one patient in the choroideremia clinical trial in
Alberta, “[The prednisone] was the hardest part
for me. I was angry and upset and after my
husband had to start a second round of it. I was
kind of thinking, ‘was this the right thing to do?’[1]
Careful consideration of the patient’s experience
throughout the clinical trial should be considered
in the planning stages to avoid miscommunication
and psychological harm.

Gene augmentation vs gene editing

Gene therapies typically employ a gene
augmentation or gene editing strategy. In the
case of gene editing, the entities introduced
into patient cells (for example, a CRISPR/Cas9
system) are designed to correct the mutation in
the endogenous copy (or copies) of the gene.
In augmentation gene therapy, the mutated/non-
functional copies of the gene are ignored, and
patient cells are supplemented with a functional
copy of the gene.[2] This strategy depends upon
sufficiently healthy residual cells being present that
can be “rescued” by the therapy. If photoreceptors
have already been lost, then alternate approaches
must be utilized. In a gene therapy technique
called “optogenetics”, non-photoreceptor cells are

made photosensitive by expressing channel
proteins. A clinical trial of the optogenetic
approach is currently recruiting patients with
retinitis pigmentosa regardless of the genetic
etiology of their condition (ClinicalTrials.gov
NCT03326336).

Clinical trials of ocular gene therapies to treat
several monogenic disorders (choroideremia, X-
linked retinoschisis, X-linked retinitis pigmentosa,
achromatopsia, and Leber hereditary optic
neuropathy) are underway across North America.
AAV is a common choice as a vector for gene
delivery. Despite initial hope that AAV vectors
would be minimally immunogenic, preliminary
results are available and adverse events have
been reported in response to the vectors in some
cases.

Routes of vector administration

The route of administration for an ocular gene
therapy vector depends on the target cells and
the tropism of the vector itself, but will generally
fall into one of two categories: intravitreal injection
or subretinal injection.[3] Wild-type AAV vector
capsids do not penetrate deeply into the retina
when administered intravitreally, but the ability
of novel AAV capsids (produced via directed
evolution) to transduce the posterior cells of the
retina is under investigation.[4, 5] An additional
barrier to successful transduction of any retinal
cells via intravitreal injection is the humoral
immune system; intravitreal injection of non-human
primates with both the AAV2 and AAV8 serotypes
was found to stimulate production of neutralizing
antibodies in subjects with and without pre-
existing immunity to the serotype.[3, 6] Conversely,
subretinal injection directly exposes the posterior
cells of the retina to the vector and evades
humoral immune surveillance; however, the retinal
detachment required to create space for vector
administration triggers microglial activation and
photoreceptor death.[7]

The remaining question of immune response

The natural triggers of immunity from the viral
vectors remain problematic in experimental
therapies.[8] In our center, in an investigator-
sponsored (Phase 1) trial of a subretinally
injected AAV gene therapy for choroideremia,

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Ocular Gene Therapy; Casey et al

one subject experienced a serious loss of
vision.[9] Unfortunately, the disease progressed
without apparent benefit in all patients. We
now question why this serious adverse event
occurred? Could it have been prevented by
an alternative vector design? Why was steroid
(given perioperatively) not sufficient to manage
inflammation? In our trial, all subjects were
treated with high-dose oral steroid (prednisone 1
mg/kg/day 3 days prior to surgery, continuing
for 21 days). The steroid was given as a
prophylactic to suppress the potential risk to
the eye of the subretinal injection. Steroid has
both local and systemic anti-inflammatory and
immunosuppressive effects; briefly, prednisone
inhibits neutrophil migration.[10] It decreases
mononuclear phagocyte chemotaxis and the
production of interleukins and TNF-α. It causes the
redistribution of CD4+ and CD8+ T-lymphocytes,
and inhibits T-lymphocyte activation, proliferation,
and lymphokine production. At high doses,
it inhibits immunoglobulin production by B-
lymphocytes.

While prophylactic steroids may aid in
regulating players of adaptive immunity such
as T-lymphocytes and B-lymphocytes, these
cells would not occupy retinal tissue unless
a severe breach had occurred in the blood–
retinal barrier. For the purposes of retinal gene
therapy, local innate immune factors are the
primary influencers of an inflammatory response
and are of therapeutic importance. To illustrate,
our choroideremia gene therapy trial utilized a
subretinal injection procedure to introduce an
AAV vector into the retina. Retinal detachment
induces local TNF-α secretion which in turn
may promote autophagy of photoreceptors by
resident microglia.[11] Even without considering
the immunological effects of the viral vector or
the diseased microenvironment of the patients’
retinas, the gene therapy procedure itself calls for
the incorporation of a local immune regulator to
prevent such adverse events.

Both a diseased microenvironment and retinal
detachment from the AAV injection “prime” the
local immune state to recognize and respond to
viral vectors more effectively. AAV viruses are
DNA-based and are thus recognized by toll-like
receptor 9 (TLR9). TLR9 stimulation in retinal
pigment epithelial cells (RPE) by CpG-DNA induces
secretion of the pro-inflammatory cytokine IL-8,

which represents the initiation of an inflammatory
cascade.[12] Preventing this TLR9-initiated
inflammatory cascade is essential when designing
an immunity regulator in AAV-based retinal gene
therapy. Our team is investigating a modified AAV
vector that blocks the dimerization and immune
signaling of TLR9. Should this vector be effective
in preventing RPE from releasing pro-inflammatory
cytokines in response to AAV treatment, we will
move from cell culture models to animal models
to confirm safety of the vector before attempting
a second choroideremia gene therapy clinical
trial.

Experimental vector gene therapies for
hereditary ocular disorders will continue
to improve, gain popular exposure, and
march toward regulatory approval. A better
understanding of the impacts of innate
immunity in the retina will improve the
safety profile of these therapies and improve
the probability of positive outcomes for
patients.

Financial Support and Sponsorship

Alberta Innovates Health Solutions, Canadian
Institutes of Health Research, Fighting Blindness
Canada.

Conflicts of Interest

There are no conflicts of interest.

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