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Review Article
Guiding Lights in Genome Editing for Inherited Retinal Disorders:
Implications for Gene and Cell Therapy

Carla Sanjurjo-Soriano1,2 and Vasiliki Kalatzis 1,2

1Inserm U1051, Institute for Neurosciences of Montpellier, Montpellier, France
2University of Montpellier, Montpellier, France

Correspondence should be addressed to Vasiliki Kalatzis; [email protected]

Received 26 January 2018; Accepted 18 April 2018; Published 8 May 2018

Academic Editor: Melissa R. Andrews

Copyright © 2018 Carla Sanjurjo-Soriano and Vasiliki Kalatzis. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.

Inherited retinal dystrophies (IRDs) are a leading cause of visual impairment in the developing world. These conditions present an
irreversible dysfunction or loss of neural retinal cells, which significantly impacts quality of life. Due to the anatomical accessibility
and immunoprivileged status of the eye, ophthalmological research has been at the forefront of innovative and advanced gene- and
cell-based therapies, both of which represent great potential as therapeutic treatments for IRD patients. However, due to a genetic
and clinical heterogeneity, certain IRDs are not candidates for these approaches. New advances in the field of genome editing using
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) have provided an
accurate and efficient way to edit the human genome and represent an appealing alternative for treating IRDs. We provide a
brief update on current gene augmentation therapies for retinal dystrophies. Furthermore, we discuss recent advances in the
field of genome editing and stem cell technologies, which together enable precise and personalized therapies for patients. Lastly,
we highlight current technological limitations and barriers that need to be overcome before this technology can become a viable
treatment option for patients.

1. Introduction

The eye, and more specifically the retina, as an extension of
the central nervous system (CNS), provides a powerful and
unique “window” to study neuronal diseases. The retina
shares anatomical and developmental characteristics with
the brain [1]. For example, it is relatively immunoprivileged
and has specialized immune responses similar to the ones
found in the brain and spinal cord [2, 3]. In addition, it is sur-
rounded by the inner blood-retinal barrier (BRB), which is
composed of the same nonfenestrated endothelial cells as
those found in the blood-brain barrier (BBB) [4]. Due to
the accessibility of the eye by modern techniques of vitreoret-
inal surgery, it is not surprising that major research and
understanding in the context of the CNS has emerged from
studies of the retina and the optic nerve [5–11]. Furthermore,
the significant compartmentalization of the eye, and specifi-
cally the retina, has allowed it to become a prototype for
the development of innovative therapies and has brought

ocular diseases to the forefront of clinical translation for
gene- and cell-based therapies. Here, we will specifically
review current progress in these therapeutic strategies for
diseases of the posterior retina (namely the neuronal pho-
toreceptor cells). Optic neuropathies affecting the anterior
retina (retinal ganglion cells (RGCs)) and optic nerve are
beyond the scope of this review.

2. The Retina

The retina is an embryonic extension of the prosencephalon
[12]. It lines the back of the eye and consists of multiple cell
layers that are responsible for the detection and processing
of visual information. The retina has a highly structured
architecture that can be divided into a posterior pigmented
monolayer and an anterior multilayered neuroretina. The
posterior layer, the retinal pigment epithelium (RPE), plays
an important role in protection (excess light absorption,
phagocytosis, water and ion transport) and support (growth

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Volume 2018, Article ID 5056279, 15 pages

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factor section, nutrient transport) of the photoreceptor layer
[13, 14]. The neuroretina is highly stratified, and it is com-
posed of three layers of specialized neurons that are intercon-
nected by two synaptic layers (Figure 1). The first layer
comprises the photosensitive rod and cone photoreceptor
cells with their characteristic outer segments, within which
the phototransduction process that follows light interaction
takes place. Light intensity dictates which photoreceptor cells
are used. In bright light, it is the centrally prevalent cones,
and in low light, it is the peripherally prevalent rods. The
photoreceptors then synapse with interneurons within the
second layer, which transmit the electrical signal arriving
from the photoreceptors to the RGCs in the third layer
[15]. The axons of the RGCs form a nerve fibre layer, which
becomes the optic nerve, and hence, the signal is transmitted
from the eye to the brain for image interpretation. The inabil-
ity to convert the light signal and transmit the electrical signal
to the brain is the primary cause of visual impairment in the
developing world. A large proportion of cases is due to dys-
function and/or loss of photoreceptors caused by a series of
risk factors including age, diabetes, and genetics [16]. The lat-
ter gives rise to a specific subset of conditions referred to as
inherited retinal dystrophies.

3. Inherited Retinal Dystrophies

Inherited retinal dystrophies (IRDs) are a genetically and
clinically heterogeneous group of neurodegenerative disor-
ders that lead to progressive visual impairment [16, 17]. They
affect approximately 1 in 2000 individuals worldwide [18].
IRDs have been associated with mutations in more than
250 genes (see, affecting
the development, function and/or survival of the photo-
receptors, and RPE [19], and with autosomal dominant,

recessive, or X-linked transmission [16]. Furthermore, com-
plex, multifactorial, and heterogeneous diseases such as
age-related macular degeneration (AMD) are also considered
retinal dystrophies.

IRDs can be divided into nonsyndromic forms, charac-
terized by an isolated retinal phenotype, or syndromic forms,
in which another organ in addition to the eye is affected.
Nonsyndromic IRDs can be further broken down into sub-
groups based on the disease progression and the region of
the retina that is affected. Firstly, progressive conditions
affecting exclusively the central retina (macula), leading to
central vision loss, are known as macular dystrophies. The
most common example is Stargardt disease with a prevalence
of 1/10000, which is due to mutations in the gene ABCA4
[20]. Secondly, progressive conditions affecting the retina
more widely can be classified depending on the type of pho-
toreceptor that degenerates initially. Rod-cone dystrophies,
where the rods are first affected, are characterized initially
by night blindness and subsequently by peripheral vision
loss; the most prevalent example (1/4000) is retinitis pigmen-
tosa (RP), caused by mutations in over 80 genes [21]. By con-
trast, in cone-rod dystrophies, the cones are first affected,
leading to decreased sharpness of visual acuity and blind
spots in the center of the visual field; ABCA4 mutations also
account for the majority of these cases [22].

When both the macula and the peripheral retina are
affected and there is a rapid retinal degeneration from birth,
the condition is known as Leber congenital amaurosis (LCA;
prevalence of 1/50000), of which 18 types are recognized. In
addition, if the retinal changes are associated with a degener-
ation of the choroid, a highly vascular, pigmented tissue
underlying the retina, these diseases are referred to as chor-
ioretinopathies. Choroideremia (CHM) is the most common
example (prevalence of 1/50000) in this group. The most




Horizontal cells

Amacrine cells
Bipolar cells

Ganglion cells






Connecting cilium

Outer segments

Figure 1: Schematic representation of the retina and the retinal cell layers. The retina is a layered structure lining the back of the eye
consisting of a pigmented layer, the RPE, and a multilayered neuroretina. The RPE is in close contact with the outer segments of the
photosensitive rod and cone cells of the neuroretina. The connecting cilium connects the photoreceptor outer segments with the cell
bodies, which constitute a layer known as the outer nuclear layer (ONL). The axons of the photoreceptors synapse with the neuronal
(bipolar, amacrine, and horizontal) cells of the inner nuclear layer (INL) via the outer plexiform layer (OPL). The axons of the INL cells in
turn synapse with the ganglion cell layer (GCL) via the inner plexiform layer (IPL). The axons of the ganglion cells converge to form the
optic nerve.

2 Neural Plasticity

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preventing the RNA polymerase and transcription factors
from transcribing genes. This strategy has been success-
fully achieved in eukaryotes and human cells [131–133].
Currently, this approach has not been applied to retinal
dystrophies, but it carries a great potential due to the mini-
mal off-target effects, which is an improvement to previous
strategies involving RNA interference [134, 135].

Ablation of the mutant allele using CRISPR/Cas9 tech-
nology is another strategy that has been used in dominant
forms for RP due to mutations in the gene encoding Rhodop-
sin (RHO). Bakondi and colleagues targeted an allele-specific
PAM sequence present only in the RhoS334mutant allele of an
RP mouse model. Following subretinal administration and
electroporation of the CRISPR components, the photorecep-
tor phenotype was rescued and visual acuity increased by
53% [136]. Similarly, Latella et al. performed a targeted
knockout of a patient-derived mutant RHO P23H minigene
in a transgenic mouse model. Subretinal electroporation of
Cas9 and two gRNA targeting the 5′ and the 3′ regions of
exon 1 resulted in reduced expression of the RHO gene
[137]. These studies carry huge promise for the use of
CRISPR/Cas systems to inactivate autosomal dominant path-
ogenic alleles in humans.

The rapid development of these technologies and the
success achieved by proof-of-concept studies in vivo are
speeding up the clinical translation of CRISPR technology.
There is currently no CRISPR-based clinical trial for eye
disease. Nonetheless, this may soon change as EDITAS
medicine appears dedicated to bringing the aforementioned
intron 26 skipping approach for CEP290 to LCA10 patients

7. Ex Vivo Gene Correction and Cell-
Based Therapy

While gene-based therapies may halt or at least slow down
the progression of the disease by targeting dysfunctional
cells, another promising approach in treating retinal dys-
trophies is stem cell-derived retinal cell transplantation.
The retina develops from the neuroectoderm, thus, like
any other CNS tissue, presents a low regeneration potential.
Therefore, IRDs caused by degeneration or loss of photore-
ceptors could potentially benefit from cell-based therapies,
which would restore a functional retina and reverse the
ocular condition.

The first evidence showing functional photoreceptor
replacement was achieved when freshly dissociated rod pho-
toreceptors were transplanted into the subretinal space [138].
However, the number of transplanted cells could not be
increased in vitro due to their postmitotic state. Thus, there
was a need to increase the number of photoreceptors for
efficient transplantation into the donor retina. Lamba and
colleagues showed that human embryonic stem cells (ESCs)
can be directed to a retinal cell fate and differentiated into
retinal precursors [139]. The transplantation of these ESC-
derived photoreceptors precursors into the subretinal space
of an LCA mouse model resulted in restoration of the light

response, establishing ESCs as a source for photoreceptor
replacement [140]. ESCs present a high proliferative, self-
renewal, and differentiation potential, which makes them
an ideal tool to study human diseases in vitro.

However, the use of ESCs is associated with controversial
and ethical considerations, thus severely impeding major
progress towards exploiting their full potential. Takahashi
et al. performed groundbreaking work in 2007, which over-
came the major limitations associated with the use of human
ESCs. Takahashi et al. demonstrated that it is possible to gen-
erate induced pluripotent stem cells (iPSCs) from adult
human fibroblasts by a reprograming process, which involves
expression of four transcription factors that revert the
somatic cells to a pluripotent state [141]. These cells have
the potential to replace patient’s tissue and represent a large
source of cells for the study of human disease [142, 143]. In
addition, iPSC-derived cells have two major advantages in
terms of cell transplantation: they avoid the ethical issues
associated with the use of embryonic or fetal tissue and they
offer the possibility of autologous transplantation avoiding
risks of immune rejection.

Both ESCs and iPSCs have been used extensively in the
area of stem cell-derived photoreceptor generation and
transplantation. Sasai and colleagues revolutionized this field
by showing that it is possible to mimic optic morphogenesis
in 3D culture using murine [144] and human [145] ESCs
and thus obtain a large source of appropriate-staged photore-
ceptor precursors. It was subsequently shown that, if present
in sufficient numbers, both ESC-derived and donor photore-
ceptor precursors could restore visual function in preclinical
retinal models [140, 146–149]. In addition, it was demon-
strated that photoreceptor precursors [150–152] as well as
functional [153] photoreceptors could also be obtained from
iPSCs. Moreover, iPSC-derived photoreceptor precursors
were transplantable and could also restore vision in preclini-
cal models [154]. Human ESCs and iPSC will continue to
have a huge impact on the study and the treatment of human
eye disease, as more optimal and standardized differentiation
protocols continue to be developed.

The coupling of iPSC and CRISPR/Cas genome-
editing technologies to repair patient-specific mutations
brings us to a new era of precise and personalized medicine
for patients. Advances have already been made for the
CRISPR/Cas-mediated correction of pathogenic mutations
causing retinal dystrophies in patient’s iPSCs. Bassuk and
colleagues were the first to demonstrate the potential of this
approach by correcting a missense mutation in RPGR
responsible for X-linked RP [155]. Burnight and colleagues
performed proof-of-concept studies for the correction of
an exonic, deep intronic, and dominant gain of function var-
iants: targeting an Alu insertion in exon 9 of MAK restored
the retinal transcript and protein, NHEJ corrected a cryptic
splice variant in CEP290-causing LCA10, and mutant
allele-specific targeting invalidated the dominant Pro23His
mutation in the RHO gene [156]. Further upstream, the
most prevalent c.2299delG mutation in the USH2A gene,
responsible for Usher syndrome type 2, was corrected in
patient’s fibroblasts using CRISPR/Cas9 and HDR [157].
These proof-of-concept studies support the development

8 Neural Plasticity

Page 9


of personalized iPSC-based transplantation therapies for
retinal disease. On a different note, CRISPR/Cas technology
in iPSCs has been used for fluorescent reporter gene knock-
in at the termination codon of the cone-rod homeobox
(Crx) gene, a photoreceptor-specific transcription factor
gene. This allows the real-time monitoring of photoreceptor
differentiation [158], demonstrating the interest of this tech-
nology also for fundamental research.

Following on from the big and promising advances,
which demonstrated that stem cell-derived photoreceptor
transplantation can restore rod- and cone-mediated vision,
recent studies demonstrated that these transplanted cells
do not integrate into nondegenerative host retinas. Instead,
postmitotic donor and host photoreceptors engage in the
transfer of cellular material, such as RNA and proteins
including Rhodopsin [149, 159–161]. The visual im-
provements observed after stem cell-derived photoreceptor
transplantation were hypothesized to be the result of
endogenous photoreceptors supplemented by donor cell-
derived proteins. More recently, it was shown that both
cell integration and cytoplasmic transfer can take place
in degenerative hosts and that the relative contributions
would depend on the local host environment [162]. Eluci-
dation of the underlying mechanisms of this cellular mate-
rial transfer could lead to novel therapeutic approaches in
introducing functional proteins into dysfunctional photo-
receptors as an alternative to gene replacement. In partic-
ular, it opens up the attractive possibility that Cas9 could
be delivered as a purified protein for genome editing of
viable photoreceptors.

The use of stem cell-derived photoreceptors is a powerful
tool for the understanding of human retinal development
and disease modeling and underlies a great potential for
developing cell transplantation therapies. Such therapies are
already underway in the clinic using hESC- [163–165] or
hiPSC-derived [166] RPE. Initially, hESC-derived RPE was
subretinally administered into AMD and Stargardt patients
as dissociated cells. These cells safely persisted over time in
the host retina and stably rescued visual acuity in a subset
of patients [164]. Just recently, an RPE patch comprising a
fully differentiated hESC-derived RPE monolayer on a
coated, synthetic basement membrane was transplanted into
AMD patients [165]. A one-year follow-up showed persis-
tence of the sheet, which was associated with increased visual
acuity and reading speed. It remains to be seen if these
improvements will be stable over time. Lastly, the first ever,
autologous transplantation for the retina was performed
using a free hiPSC-derived RPE monolayer [166]. A one-
year follow-up showed that the transplantation was safe
and no immune response was provoked even in the absence
of immunosuppression. This provides hope for the future
autologous transplantation of genome-edited retinal cells in
patients. Nonetheless, further work is required to establish
robust and reproducible protocols for the generation of
iPSC-derived photoreceptors. In addition, if such cells are
transplanted following gene mutation repair, stringent
quality control of the iPSCs before and after gene correction
is extremely important. Furthermore, a detailed screening
for possible off-target effects triggered by CRISPR/Cas

has to be performed prior to transplantation into the
diseased host retina.

8. Future Challenges and Perspectives

The eye, more specifically the posterior retina, has proven
to be a powerful model for the development of pioneer
therapies, which could later be applied to other parts of
the CNS. Despite the current success achieved by researchers
and the relative ease and precise manipulation of the genome
using the CRISPR/Cas system, improvements are being
made. These are focused on the development of more effi-
cient delivery methods, the identification and understand-
ing of the off-target events, and increasing the efficiency
of mutation correction. All these matters should be care-
fully addressed before this strategy can be safely applied
in the clinic.

Potential delivery methods of the CRISPR/Cas compo-
nents can be diverse. For an in vivo application, the ideal
vehicle would be an AAV vector. The limitation of this
method, in addition to size restrictions, is the constitutive
expression of the Cas9 protein in the host organism,
which increases the risk of unwanted off-target events
in the genome [98–100]. The use of Cas9 RNP has been
shown to be effective in vivo for reducing off-target events
[101, 102, 167], although to our knowledge there has not
been a study directly comparing the off-target effects of a
given gRNA by AAV or RNP delivery. Thus, future research
is needed in order to elucidate the most effective way, with
high on-target activity and null off-target activity, to deliver
CRISPR/Cas components in vivo.

A variety of methods aimed at testing for off-target muta-
tions have been developed [168–170]. These methods are
based on algorithms to computationally test homologous
regions in the genome. However, currently, there is no gold
standard, and it is not yet clear if Cas9 has the potential to
alter other nonhomologous regions in the genome. Some
studies have performed whole exome sequencing (WES) in
CRISPR-treated cells and organisms [171, 172], providing
an accurate and comprehensive way of testing off-target
mutations. Such approaches should be taken into consider-
ation following ex vivo gene correction in view of future
transplantation into the patient.

In addition to improving the understanding of the off-
target effects created by Cas9, much effort has focused on
developing methods to enhance genome-editing efficiency.
In cases where gene correction is required, the HDR repair
pathway is needed, and this is incompatible with postmi-
totic photoreceptor targets. Exciting new developments in
HDR-independent base-editing strategies have shown prom-
ise for gene correction in postmitotic cells. In these cases,
Cas9 is fused to a cytidine deaminase to create a base-editor
tool at the specific genome target [173, 174], thus circum-
venting the need for cell division. In addition, as mentioned
above, the HITI approach also carries a great promise for
precise gene correction in postmitotic cells by using the
NHEJ pathway [127].

Overall, the future looks bright for the use of CRISPR/
Cas genome editing in ophthalmology, and it is likely that

9Neural Plasticity

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[153] X. Zhong, C. Gutierrez, T. Xue et al., “Generation of three-
dimensional retinal tissue with functional photoreceptors
from human iPSCs,” Nature Communications, vol. 5,
p. 4047, 2014.

[154] B. A. Tucker, I. H. Park, S. D. Qi et al., “Transplantation of
adult mouse iPS cell-derived photoreceptor precursors
restores retinal structure and function in degenerative mice,”
PLoS One, vol. 6, no. 4, article e18992, 2011.

[155] A. G. Bassuk, A. Zheng, Y. Li, S. H. Tsang, and V. B. Mahajan,
“Precision medicine: genetic repair of retinitis pigmentosa in
patient-derived stem cells,” Scientific Reports, vol. 6, no. 1,
article 19969, 2016.

[156] E. R. Burnight, M. Gupta, L. A. Wiley et al., “Using CRISPR-
Cas9 to generate gene-corrected autologous iPSCs for the
treatment of inherited retinal degeneration,”Molecular Ther-
apy, vol. 25, no. 9, pp. 1999–2013, 2017.

[157] C. Fuster-García, G. García-García, E. González-Romero
et al., “USH2A gene editing using the CRISPR system,”
Molecular Therapy Nucleic Acids, vol. 8, pp. 529–541, 2017.

[158] K. Homma, S. Usui, and M. Kaneda, “Knock-in strategy at 3′
-end of Crx gene by CRISPR/Cas9 system shows the gene
expression profiles during human photoreceptor differentia-
tion,” Genes to Cells, vol. 22, no. 3, pp. 250–264, 2017.

[159] R. A. Pearson, A. Gonzalez-Cordero, E. L. West et al., “Donor
and host photoreceptors engage in material transfer following
transplantation of post-mitotic photoreceptor precursors,”
Nature Communications, vol. 7, article 13029, 2016.

[160] T. Santos-Ferreira, S. Llonch, O. Borsch, K. Postel, J. Haas,
and M. Ader, “Retinal transplantation of photoreceptors
results in donor–host cytoplasmic exchange,” Nature Com-
munications, vol. 7, article 13028, 2016.

[161] S. Decembrini, C. Martin, F. Sennlaub et al., “Cone genesis
tracing by the Chrnb4-EGFP mouse line: evidences of cellular
material fusion after cone precursor transplantation,”Molec-
ular Therapy, vol. 25, no. 3, pp. 634–653, 2017.

[162] P. V. Waldron, F. di Marco, K. Kruczek et al., “Transplanted
donor- or stem cell-derived cone photoreceptors can both
integrate and undergo material transfer in an environment-
dependent manner,” Stem Cell Reports, vol. 10, no. 2,
pp. 406–421, 2018.

[163] S. D. Schwartz, J. P. Hubschman, G. Heilwell et al., “Embry-
onic stem cell trials for macular degeneration: a preliminary
report,” The Lancet, vol. 379, no. 9817, pp. 713–720, 2012.

[164] S. D. Schwartz, C. D. Regillo, B. L. Lam et al., “Human embry-
onic stem cell-derived retinal pigment epithelium in patients
with age-related macular degeneration and Stargardt’s macu-
lar dystrophy: follow-up of two open-label phase 1/2 studies,”
The Lancet, vol. 385, no. 9967, pp. 509–516, 2015.

[165] L. da Cruz, K. Fynes, O. Georgiadis et al., “Phase 1 clinical
study of an embryonic stem cell-derived retinal pigment epi-
thelium patch in age-related macular degeneration,” Nature
Biotechnology, vol. 36, no. 4, pp. 328–337, 2018.

[166] M. Mandai, A. Watanabe, Y. Kurimoto et al., “Autologous
induced stem-cell–derived retinal cells for macular degenera-
tion,” The New England Journal of Medicine, vol. 376, no. 11,
pp. 1038–1046, 2017.

[167] M. A. DeWitt, J. E. Corn, and D. Carroll, “Genome editing via
delivery of Cas9 ribonucleoprotein,” Methods, vol. 121-122,
pp. 9–15, 2017.

[168] X. H. Zhang, L. Y. Tee, X. G. Wang, Q. S. Huang, and S. H.
Yang, “Off-target effects in CRISPR/Cas9-mediated genome

engineering,” Molecular Therapy Nucleic Acids, vol. 4, article
e264, 2015.

[169] R. Peng, G. Lin, and J. Li, “Potential pitfalls of CRISPR/Cas9-
mediated genome editing,” The FEBS Journal, vol. 283, no. 7,
pp. 1218–1231, 2016.

[170] J. K. Yee, “Off-target effects of engineered nucleases,” The
FEBS Journal, vol. 283, no. 17, pp. 3239–3248, 2016.

[171] K. Nakajima, A. A. Kazuno, J. Kelsoe, M. Nakanishi,
T. Takumi, and T. Kato, “Exome sequencing in the knockin
mice generated using the CRISPR/Cas system,” Scientific
Reports, vol. 6, no. 1, article 34703, 2016.

[172] S. W. Cho, S. Kim, Y. Kim et al., “Analysis of off-target effects
of CRISPR/Cas-derived RNA-guided endonucleases and
nickases,”Genome Research, vol. 24, no. 1, pp. 132–141, 2014.

[173] A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, and D. R.
Liu, “Programmable editing of a target base in genomic
DNA without double-stranded DNA cleavage,” Nature,
vol. 533, no. 7603, pp. 420–424, 2016.

[174] N. M. Gaudelli, A. C. Komor, H. A. Rees et al., “Programma-
ble base editing of A•T to G•C in genomic DNA without
DNA cleavage,” Nature, vol. 551, no. 7681, pp. 464–471,

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