Leber congenital amaurosis (LCA) is a group of monogenic inherited retinal degenerations
that typically show early onset and severe visual dysfunction. In addition, there
is a natural history of progressive loss of photoreceptors and associated further
loss of vision. The therapeutic goal of slowing the natural history of degenerative
disease has a long history of effort and deserves our attention, but it is imperative
to have realistic timelines and careful protocols that define how efficacy will be
measured over many years. The therapeutic goal of improving vision is easier to detect
over a shorter period of observation. However, modern techniques of noninvasive examination
in LCA have demonstrated that only a subset of patients can be predicted to have potential
for improvement of vision, given safe and effective therapies. Discussed herein are
two LCA subtypes, the ciliopathy of CEP290-LCA and the phototransduction defect of
GUCY2D-LCA, that show a common potential for improvement of vision despite differences
in molecular mechanism.
Mutations in several hundred genes are now known to cause inherited retinal diseases
(IRDs).1–4 IRDs represent a highly heterogeneous group of disorders that have one
common element: abnormal visual function originating at the level of retinal photoreceptors.
A subset of IRDs comprises syndromic diseases, whereas the majority are nonsyndromic
and affect only the retina even when the gene is expressed ubiquitously. The source
of photoreceptor dysfunction can be due to maldevelopment of cells,5,6 a defect in
the neighboring retinal pigment epithelium (RPE) cells,7,8 progressive loss of cells
to neurodegeneration,9 a variety of other pathophysiological mechanisms,10 or a combination
thereof. Until a decade ago, IRDs were treated mainly with nutrient supplements aimed
to slow the disease.11 Then, converging information from molecular and retinal biology,
animal models, human phenotyping, and therapeutic tools reached a critical mass,12
–19 and led to the first successful proof of concept of a gene-based treatment of
an IRD caused by RPE65 mutations.20
–23 Extensive research followed to describe the extent and the source of improvement
of visual function, durability of the treatment, and the effects on the rate of photoreceptor
degeneration.24
–30 Recent approval of this gene therapy approach for marketing in the United States
and Europe31 has generated greater interest in the development of treatments for other
IRDs.
There is no one-size-fits-all approach to gene-based treatments for IRDs. Therapeutic
directions for different IRDs need to be aligned with the underlying molecular pathophysiology,
with the tools available for their delivery, and with consideration of the recipient
retinal cells expected to be retained at the time of the intervention. For example,
larger genes cannot be packaged into some viral vectors,32 or rod photoreceptor-based
treatments would not be appropriate for adult patients with Class A rhodopsin mutations
who have only cones remaining.33 Outcome measures also need to be optimized to the
expected magnitude and timing of the efficacy signal. For example, evidence for successful
slowing of disease progression may take years to detect34,35 but improvements in vision
may occur in days to weeks.22,36
Among the more severe IRDs are those clinically classified as LCA. LCA manifests vision
loss that usually occurs congenitally or in early infancy. There is nystagmus (involuntary
eye movements), and a deceptively benign fundus appearance at early stages, but the
disease expression tends to be indistinguishable from that of other IRDs at later
stages. Abnormal electroretinograms localize the vision defect to the outer retina,
at the photoreceptors. The attraction of some forms of LCA as a target for therapy
rests not only in the severity of the vision loss but also in the key fact that improvement
of visual function is potentially achievable because there is evidence of dissociation
of function and structure.16 The first form of LCA with sufficient proof of concept
research, understanding of molecular mechanism, and detailed pretreatment human disease
characterization indicating structural preservation but severe functional losses was
the RPE disease with a defective retinoid cycle: RPE65-LCA.25 The improved vision
in RPE65-LCA patients post therapy in three clinical trials conducted independently
but almost simultaneously, is in contrast to the results in another RPE-based severe
and early-onset IRD caused by mutations in MERTK. Despite longstanding rodent proof
of concept research and understanding of mechanism, there was limited characterization
of the human disease and no published demonstration of structure–function dissociation.
A clinical trial protocol mimicking that of the RPE65-LCA trials in MERTK patients
failed to achieve the efficacy results of RPE65-LCA.37
Two photoreceptor diseases within the clinical category of LCA, CEP290-LCA and GUCY2D-LCA,
have been shown to have a dissociation of structure and function in affected humans
and thus are good candidates for appropriate vision improving therapies.38
–45
CEP290-LCA
Photoreceptor Cilium and LCA-Ciliopathies
Rod and cone photoreceptors are specialized cells for phototransduction, and this
process converts light into neural signals and vision. Photoreceptors have four major
compartments: the outer and inner segments, the cell body, and the synaptic terminal
(Fig. 1A). As part of their specialization, the photoreceptors contain one of the
longest sensory cilia known in mammalian cells, extending across the inner and outer
segments. Between the inner and outer segments of photoreceptors is the ciliary transition
zone, also called the connecting cilium, which can be thought of as a two-way highway
for the trafficking of all the proteins to and from the outer segment, where phototransduction
takes place, and a gate that allows appropriate compartmentalization of proteins (Figs.
1B, 1C).
Figure 1
CEP290 expression in rod and cone photoreceptors. (A) Schematic of a rod photoreceptor,
showing specialized domains of the cell. (B) Enlargement of the rod photoreceptor
transition zone showing the structural and functional domains in which most ciliary
proteins are expressed: axoneme (green), connecting cilium/transition zone (CC/TZ;
orange), basal body (BB; purple), periciliary complex or ciliary pocket (PCC/CP; red).
(C) Cross section through the CC/TZ showing the relationship between the microtubules
of the cilium and the inner segment, via the PCC/CP. (A–C) Reprinted and modified
with permission from Rachel RA, Li T, Swaroop A. Photoreceptor sensory cilia and ciliopathies:
focus on CEP290, RPGR and their interacting proteins. Cilia. 2012;1:22. © 2012 The
Authors. Published by BioMed Central, Ltd. (D) Three-dimensional representation of
the transition zone and adjacent domains. Possible positions of rod-like coiled-coil
domain proteins such as CEP290, which localize to the region of the Y-linkers between
the plasma membrane and the microtubule ring. Reprinted and modified with permission
of Rachel RA, Yamamoto EA, Dewanjee MK, et al. CEP290 alleles in mice disrupt tissue-specific
cilia biogenesis and recapitulate features of syndromic ciliopathies. Hum Mol Genet.
2015;24:3775–3791. © 2015 The Authors. Published by Oxford University Press. (E) Immunofluorescence
staining of CEP290 in macular cones of monkey retina. Sections stained with CEP290
(green) and cone-specific marker PNA (red) indicate colocalization (Merge; arrows).
DAPI (blue) used to stain the nuclei. Scale bar: 10 μm. Reprinted with permission
from Cideciyan AV, Aleman TS, Jacobson SG, et al. Centrosomal-ciliary gene CEP290/NPHP6
mutations result in blindness with unexpected sparing of photoreceptors and visual
brain: implications for therapy of Leber congenital amaurosis. Hum Mutat. 2007;28:1074–1083.
© 2007 John Wiley & Sons, Inc. Published by Wiley-Liss, Inc.
It has been long hypothesized that the connecting cilium may be a primary site of
disease in some inherited retinopathies,46 and now it is known that large numbers
of IRD genes are expressed at the connecting cilium.47,48 Indeed, at least one-third
of the molecular pathways known to be associated with syndromic or nonsyndromic forms
of LCA are thought to be ciliopathies.49
–51
CEP290 (centrosomal protein, 290 kD) gene encodes a large protein that is located
at the transition zone of the rod photoreceptors52,53 (Fig. 1D), and mutations in
CEP290 are among the most common genetic causes of LCA.54
–56 CEP290 is also expressed in primate cone photoreceptors (Fig. 1E).
Visual Consequences
LCA is generally considered more severe than other IRDs such as retinitis pigmentosa
(RP). However, even within LCA, different molecular subtypes can show substantial
differences in severity. Defining severe vision loss as a visual acuity of counting
fingers (CF) or worse in the best-seeing eye, 62% to 89% of patients with CEP290-LCA
have severe vision loss,38,39,57
–61 whereas this proportion is closer to 10% in RPE65-LCA.57,62,63 Importantly, many
CEP290-LCA patients report very low level of vision from as early as they can remember,
implying a congenital lack of visual functioning. Consistent with congenital or very
early-onset vision loss are the large hyperopic refractive errors in CEP290-LCA38,39,58,60
suggesting problems with emmetropization during development.
Although visual acuity is useful and understandable to differentiate among the visual
abilities of patients, it does not provide a direct measure of the primary function
of photoreceptors—to signal light levels. Sensitivity to light can be quantified for
rods and cones by determining the dimmest lights a subject can detect. Standard perimeters
use this approach to understand the retinotopic distribution of light sensitivity
in eyes fixating to a steady reference. However, oculomotor instability of LCA subjects
requires a fixation-independent approach to evaluating light sensitivity, which is
achieved with full-field stimulus testing (FST).64,65 In FST each stimulus is presented
across the full visual field and thus it does not require a stable gaze. Use of two
colors and dark-adapted eyes takes advantage of the spectral separation between rods
and cones to understand photoreceptor mediation driving the light sensitivity measured.
CEP290-LCA patients tend to show more than 4 log units of sensitivity loss.35,39,66
Chromatic sensitivity differences support mediation by long-/middle-wavelength (L/M)
sensitive cones in the great majority of the patients, but there can be exceptions.
Oculomotor Control and Fixation
CEP290-LCA patients demonstrate a wide spectrum of oculomotor abnormalities. On the
mild end of this spectrum are eyes with retained visual acuity, fixation, and small-amplitude
(fine) nystagmus. The severe end of the spectrum includes eyes with congenital lack
of light perception, demonstrating wandering eye movements, no ability to hold the
eye in primary gaze, and no fixation.35 We recently developed a video recording protocol
using a confocal scanning laser ophthalmoscope (Spectralis HRA; Heidelberg Engineering,
Heidelberg, Germany) commonly used for retinal imaging.35 The oculomotor control and
instability (OCI) protocol uses instantaneous distance between the center of the pupil
and a stationary reference such as the medial canthus to quantify two parameters over
recording epochs of 30 seconds: the offset from primary gaze, and the extent of oculomotor
instability (Figs. 2A, 2B). When performed in a dark room with and without a fixation
light, OCI results allow distinguishing between open-loop and close-loop conditions,
and thus determine any changes to the oculomotor system that may be driven by visual
input.
Figure 2
Spectrum of oculomotor features in CEP290-LCA. (A) Upper: Schematic representation
of the coordinate system centered at the medial (nasal) canthus and the center of
pupil (white cross) at primary gaze. Lower: Individual data from left (LE) and right
(RE) eyes of all normal subjects at primary gaze. Mean value is also shown (circle).
(B) Schematic representation of eyes fixating 30° eccentric along the four cardinal
directions, and relative offsets of the center of pupil measured from the primary
gaze locus. (C) Chart records showing the radial offset of the center of pupil from
the mean normal primary gaze locus (thick gray line) during a 30-second-long recording
epoch in a representative normal subject and two CEP290-LCA eyes. Two records shown
are with (right column) and without (left column) fixation. (D) Oculomotor instability
plotted against mean gaze offset in individual CEP290-LCA eyes (triangles; n = 32
eyes of 16 patients) recorded with and without fixation. Equivalent results from normal
eyes are also shown (gray circles). Gray lines demarcate the upper (mean + 2 SD) limits
of normal for each parameter. Reprinted with permission from Jacobson SG, Cideciyan
AV, Sumaroka A, et al. Outcome measures for clinical trials of Leber congenital amaurosis
caused by the intronic mutation in the CEP290 gene. Invest Ophthalmol Vis Sci. 2017;58:2609–2622.
© 2017 The Authors. Published by ARVO.
A normal eye in primary (straight-ahead) gaze corresponds to a pupil center position
that is on average 13.7 mm temporal and 3.3 mm superior with respect to the medial
canthus (Fig. 2A). Excursions of 30° visual angle from primary gaze along the four
cardinal meridians result in approximately 4-mm movements of the center of the pupil
away from the center (Fig. 2B). To a first approximation, the location of the center
of the pupil can thus be used to quantify the gaze position and its stability over
time. As demonstrated in a representative subject, normal eyes tend to be very stable
with or without fixation (Fig. 2C, upper traces). Individual CEP290-LCA patients can
have reliable control of gaze position with small-amplitude nystagmus (Fig. 2C, middle
traces), or others may have complete lack of oculomotor control with wandering eyes
(Fig. 2C, lower traces). Summary of a cohort of CEP290-LCA eyes shows a range of oculomotor
instability from 0.3 to 3.5 mm without fixation and 0.2 to 3.6 mm with fixation; similarly,
mean gaze offset ranged from 0.6 to 5.5 mm without fixation and 0.2 to 6.2 mm with
fixation (Fig. 2D).
Photoreceptor Structure
Retinal neurons and glia are laminated into three nuclear layers and two intervening
synaptic layers; distal retina contains carefully aligned photoreceptor outer segments
interdigitating with RPE apical processes. Many of the cellular and subcellular retinal
features demonstrate natural differences in how much they reflect infrared light,
and these differences are imaged by optical coherence tomography (OCT).67,68 More
than a decade ago using lower resolution and slower time-domain OCT systems, we showed
that CEP290-LCA patients with severe vision loss tend to retain macular photoreceptors.38
Further studies using higher resolution and faster spectral-domain (SD) OCT systems
provided greater information and hypotheses on retinal structure that were tested
in relevant animal models.39
–43 More recently we have used a clinical ultrahigh resolution (UHR) SDOCT system
(Bi-μ; Kowa Company, Ltd., Tokyo, Japan) to better understand microscopic features
in CEP290-LCA retinas (Fig. 3).
Figure 3
Retained photoreceptor nuclei with abnormal segments in CEP290-LCA. (A) OCT scans
along the horizontal meridian through the fovea in a normal subject, and a CEP290-LCA
patient. Images were obtained with a clinical ultrahigh resolution SDOCT system (Bi-μ;
Kowa Company, Ltd.). Hyposcattering layer corresponding to the ONL is shown. Inset
upper right shows location of scan. Yellow boxes outline foveal and perifoveal regions
shown in (B, C). (B, C) Magnified views of the outer retina at foveal and temporal
perifoveal locations demonstrating differences in the layers distal to the ONL. Overlaid
are the longitudinal reflectivity profiles (LRPs). Hyperscattering signals highlighted
as follows: green, ELM; yellow, IS/OS, near the junction of inner and outer segments;
orange, COST, near the interface of cone outer segment tips and RPE contact cylinder
(also called the interdigitation zone); cyan, ROST, near the interface between rod
outer segment tips and RPE apical processes; brown, RPE, near the RPE cell bodies;
and black, BrM, Bruch membrane. Figure courtesy of Alexander Sumaroka (Scheie Eye
Institute, University of Pennsylvania).
UHR SDOCT of the normal human retina along the horizontal meridian crossing the fovea
shows exquisite micron-scale detail of cellular and subcellular structures. Most prominent
across the center of the scan is a hyposcattering (dark) band that corresponds to
the outer nuclear layer (ONL) containing the nuclei of rod and cone photoreceptors
and the Henle fiber layer (Fig. 3A, upper). However, it is important to note that
the cellular constituents of the ONL change with eccentricity from the fovea. The
normal foveola, referring to the ∼1° diameter center of the fovea, consists of only
cone photoreceptors whereas the extrafoveal retina is mostly rods. The great majority
of CEP290-LCA patients retain a substantial ONL in the central macular region that
thins with eccentricity. The UHR SDOCT from a 10-year-old CEP290-LCA patient with
bare light perception vision illustrates a typical scan (Fig. 3A, lower). The ONL
has normal thickness at the cone-rich foveola, which suggests near-normal density
of cone photoreceptor nuclei. Between the fovea and ∼6° eccentricity, the ONL thins
steeply and asymptotes to a very thin (∼10 μm) layer with detectable outer plexiform
layer (OPL) and external limiting membrane (ELM) boundaries. This layer likely represents
a single row of cone nuclei and lacking all rod nuclei.39 Existence of normal foveal
photoreceptors despite severe reduction of vision suggests a major dissociation of
function and structure reminiscent of the RPE65 form of LCA.16 Quantitative comparison
of light sensitivity and retinal structure in CEP290-LCA patients has provided direct
evidence of the dissociation.43
Retained outer retinal photoreceptor nuclei are necessary but not sufficient to drive
retinal visual function: Also needed are outer segments for phototransduction and
inner segments for energy production. Magnified OCT images show at least four foveal
and at least five extrafoveal hyperscattering peaks distal to the ELM where inner
and outer segments and RPE processes would be expected to reside in normal eyes (Figs.
3B, 3C). These hyperscattering signals are thought to originate from histologic layers
near the junction between inner and outer segments (IS/OS, also called inner segment
ellipsoid zone), near the interface between cone outer segment (COS) tips and RPE
contact cylinder (also called the interdigitation zone, IZ), near the interface between
rod outer segment tips (ROST) and RPE apical processes, near the RPE cell bodies,
and at the Bruch membrane (BrM).67
–71 Of note, distinct ROST, RPE, and BrM peaks apparent in normal extrafoveal scans
(Fig. 3C, left) are often amalgamated into a single peak in lower-resolution clinical
images.72
At the CEP290-LCA fovea, the ELM to BrM distance is approximately half of normal and
lacks the normal layering (Fig. 3B). There can be a hyperscattering layer likely corresponding
to a widened IS/OS peak with substantially shortened distance to the ELM suggesting
shortened inner segment lengths. Instead of a hyperscattering COS tips (COST) peak,
CEP290-LCA foveas have a wide hyposcattering layer that likely corresponds to misshapen
OS or OS debris or both. In the perifoveal region, there are detectable OPL and ELM
signals suggesting a retained ONL layer that is less than one-third the normal thickness.
IS/OS, ROST, and COST peaks are not detectable in perifoveal scans of CEP290-LCA (Fig.
3C).
Disease Progression
Previously we showed that rod photoreceptors develop in CEP290-LCA but rapidly degenerate
postnatally with some patients retaining midperipheral rods in the first decade of
life.39 By the second decade of life, most patients retain central cones devoid of
rods. Considering the longstanding hypothesis that cones require rod-derived viability
factors for their survival,73 we recently estimated the rate of degeneration of cone
photoreceptors in CEP290-LCA.35 OCTs from 20 patients across five decades of life
were quantified cross-sectionally. In a subset of seven patients, longitudinal recordings
performed over nearly a decade were analyzed. The ONL thickness at the fovea was normal
or hypernormal in all patients but one (Fig. 4B). Longitudinally there were either
no detectable changes or there was some mild thinning. Combining the cross-sectional
and longitudinal results, and accounting for the patient with foveal degeneration,
foveal ONL thickness tended to decrease at an average rate of 1.3 μm/year (Fig. 4B,
gray regression line). When the patient with foveal degeneration was excluded, the
average rate of foveal ONL loss was 0.7 μm/year.
Figure 4
Slow rate of cone photoreceptor degeneration in CEP290-LCA. (A) Horizontal OCT from
a CEP290-LCA patient (left) demonstrating the foveal ONL thickness and ONL extent
measures. Near-infrared autofluorescence imaging (right) demonstrating preserved central
macular region of RPE melanization. (B, C) Quantitation of foveal ONL thickness (B),
and ONL extent from fovea in nasal and temporal directions (C) in a group of CEP290-LCA
patients evaluated cross-sectionally at different ages. Also shown are a subset of
patients with longitudinal data (connected symbols). Linear regressions (thick gray
line) fit to all data. Redrawn from data in Jacobson SG, Cideciyan AV, Sumaroka A,
et al. Outcome measures for clinical trials of Leber congenital amaurosis caused by
the intronic mutation in the CEP290 gene. Invest Ophthalmol Vis Sci. 2017;58:2609–2622.
© 2017 The Authors. Published by ARVO.
The elliptical region of central macular preservation in CEP290-LCA slowly constricts
over time, and this is demonstrated by quantifying the nasal and temporal extents
of ONL retention (Fig. 4C). Analysis of the central elliptical region extent of melanized
RPE with near-infrared reduced illuminance autofluorescence imaging (NIR-RAFI) showed
a similar slow constriction over decades.35 These results suggest a very wide window
over which the dysfunctional CEP290-mutant cones are amenable to vision improvement
treatments. However, evaluation of changes to natural history of disease would be
expected to be challenging.
Postreceptoral Structure Along the Retinocortical Pathway
Connectivity between the outer retina and the visual cortex is necessary for patients
to perceive the consequences of efficacious treatment of their cone photoreceptors.
To understand whether the pathways are available to signal photoreceptor signals to
higher vision centers, we evaluated proximal structures. Within the retina, cells
immediately postsynaptic to photoreceptors are located in the inner nuclear layer
(INL), which had normal or hypernormal thickness (Figs. 5A, 5B). Tertiary neurons
are located in the ganglion cell layer (GCL), which also had normal or hypernormal
thickness (Figs. 5A, 5B). Axons of ganglion cells are located in the retinal nerve
fiber layer (RNFL), which was also normal or hypernormal in thickness (Figs. 5A, 5B).
Thickening of inner retinal layers is thought to represent retinal remodeling38 and
was most prominent in the perifoveal retinal areas of CEP290-LCA with little evidence
of photoreceptors remaining. The central region with the retained cone photoreceptors
tended to have normal or near-normal inner retinal structure.
Figure 5
Postreceptoral structures along the retinocortical pathway in CEP290-LCA. (A) Left,
OCT scans along the vertical meridian in a normal subject and a CEP290-LCA patient.
ONL is highlighted blue, INL is highlighted purple, and GCL is highlighted orange.
(B) Quantitation of the three nuclear layer and the RNFL thickness in 6 CEP290-LCA
patients compared with normal results (shaded areas; mean ± 2 SD). (C) Optic nerve
anatomy. A normal-appearing optic chiasm (arrowhead) observed on T1 imaging. High-resolution
T2-weighted axial and coronal images were obtained through the optic nerves. The position
of the coronal slice displayed is indicated by the dashed line on the axial image.
The cross-sectional diameter of the interpial optic nerve (arrows) was estimated at
three positions along each nerve, and the average diameter is within the range of
normal (plot). (D) Whole-brain morphometric analysis. The T1-weighted anatomic images
from CEP290-LCA and controls were warped to a representative template (top row). The
(log) determinant of the Jacobian matrix calculated during warping for each subject
(bottom row) indexes the degree to which cerebral tissue is smaller or larger than
the template image. No significant deviation from control measures was seen in two
CEP290-LCA patients. A focused analysis was conducted within occipital lobe white
matter (red on the registered anatomy). The z-position (mm) of each axial slice relative
to the anterior commissure is indicated. The average (log) Jacobian measure within
the occipital white matter for CEP290-LCA and normal subjects indicates no differences
(plot). (C, D) Reprinted with permission from Cideciyan AV, Aleman TS, Jacobson SG,
et al. Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected
sparing of photoreceptors and visual brain: implications for therapy of Leber congenital
amaurosis. Hum Mutat. 2007;28:1074–1083. © 2007 John Wiley & Sons, Inc. Published
by Wiley-Liss, Inc.
What about postretinal structures? In two patients with CEP290-LCA, we had the opportunity
to make measurements. Interpial optic nerve diameters were normal (Fig. 5C), and whole-brain
morphometric analysis found no significant deviations from normal cortical or subcortical
anatomy (Fig. 5D). Retinal and postretinal anatomy taken together (Figs. 3–5) allows
us to conclude that central macular cone photoreceptors in CEP290-LCA retain sufficient
pathways to potentially carry vision signals from cone photoreceptors to the visual
cortex upon administration of a successful therapy.
Function of Postreceptoral Circuits in Patients Lacking Perception
A large subset of CEP290-LCA demonstrates severe (often congenital) lack of visual
perception. Prerequisites to any treatment approaches in these patients must include
evidence that the retained postreceptoral structures (described above) demonstrate
function. For this, we took advantage of the pupillary light reflex (PLR). PLR is
normally driven by a combination of four outer retinal photoreceptors and by melanopsin-containing
intrinsically photosensitive retinal ganglion cells (ipRGCs) depending on the stimulus
used, adaptation conditions, and ambient light levels.74,75 When outer retinal photoreceptors
are genetically, pharmacologically, or spectrally silenced, PLR is dominated by the
ipRGC function (Fig. 6). Using standard stimuli, many CEP290-LCA patients show no
detectable PLR (Figs. 6A–C). However, higher stimulus strengths allow recording of
robust signals (Figs. 6D, 6E), which suggest that at least some postreceptoral circuits
are functional. Whether this functionality extends to the visual cortex in patients
with congenital lack of vision remains to be determined.
Figure 6
Pupillary light reflexes in CEP290-LCA. (A–C) Dynamics of pupil constriction in the
dark to a 0.1-second-duration achromatic bright standard stimulus (1.5 log scot-cd.s.m−2)
in a representative normal (A), CEP290-LCA patients grouped into those with detectable
responses (B), and those without (C). (A–C) Modified from Jacobson SG, Cideciyan AV,
Sumaroka A, et al. Outcome measures for clinical trials of Leber congenital amaurosis
caused by the intronic mutation in the CEP290 gene. Invest Ophthalmol Vis Sci. 2017;58:2609–2622.
© 2017 The Authors. Published by ARVO. (D) Use of higher stimulus luminance range
in two of the eyes with no pupillary response to standard stimuli. Notably, 0.9 and
1.9 log scot-cd.s.m−2 stimuli do not evoke responses, whereas 2.9 log scot-cd.s.m−2
stimuli evoke definite responses that are smaller and slower than normal. Similarity
of responses with 0.1- and 1-second-long stimuli suggests reciprocity between stimulus
luminance and duration. (D) Modified from Charng J, Jacobson SG, Heon E, et al. Pupillary
light reflexes in severe photoreceptor blindness isolate the melanopic component of
intrinsically photosensitive retinal ganglion cells. Invest Ophthalmol Vis Sci. 2017;58:3215–3224.
© 2017 The Authors. Published by ARVO.
Treatment Approaches to CEP290-LCA
The most promising approaches to treating monogenic IRDs to date are gene augmentation
therapies using AAV vectors.25,76 However, the full-length CEP290 gene is too large
for the AAV capacity and thus alternative avenues are required for the treatment of
CEP290-LCA. Alternatives include gene editing,77,78 augmentation with gene fragments,79
–81 lentiviral vectors with larger packaging capacity,82 or splitting the transgene
across two separate AAV vectors.83 Antisense gene therapy is a promising therapeutic
approach that takes advantage of a frequently occurring intronic mutation (c.2991+1655A>G)
that creates a splice donor site that permits a cryptic exon insertion and leads to
premature termination of protein synthesis.55 Antisense RNA oligonucleotides (AONs,
or ASOs) are designed to target the pseudoexon region to increase normal protein expression.84
–86 A phase I/IIa clinical trial with such an AON is ongoing, and the current authors
are investigators at one of the sites of this trial (NCT03140969 Clinicaltrials.gov).
Preliminary results support an acceptable safety profile and a potential for improvement
of visual acuity and light sensitivity.87
GUCY2D-LCA
Phototransduction and LCA1
Phototransduction abnormalities have long been associated with IRDs,88 and mutations
in genes encoding phototransduction proteins are now known causes of inherited retinal
dysfunction and degeneration.1,2,89 The gene GUCY2D encodes retinal guanylyl cyclase
(retGC1), which modulates phototransduction in rods and cones. RetGC1 is expressed
in both cones and rods as a 120-kDa membrane protein that is responsible for the resynthesis
of cyclic guanosine monophosphate (cGMP) required for the recovery of the dark-adapted
state of photoreceptors after phototransduction (Fig. 7). Autosomal recessive mutations
in GUCY2D lead to LCA190 likely due to dysregulation of guanylyl cyclase causing an
equivalent state to chronic light exposure of photoreceptors.91
Figure 7
Phototransduction and GUCY2D. Absorption of light by rhodopsin in the rod photoreceptor
outer segment activates rhodopsin and starts the cascade of reactions that successively
include activation of transducin and cGMP phosphodiesterase (PDE), which hydrolyzes
cGMP. Reduction in the concentration of cGMP leads to closure of cGMP-gated channels
(CNG). The recovery response occurs as there is continued decrease of intracellular
Ca2+ and activation of guanylyl cyclase (retGC) by GCAP (guanylyl cyclase activating
protein). This replenishes cGMP and causes reopening of CNG. GUCY2D, the gene encoding
retGC, is key to response recovery. Redrawn and reprinted with permission from Boye
SE. Insights gained from gene therapy in animal models of retGC1 deficiency. Front
Mol Neurosci. 2014:7:43. © 2014 Boye.
Visual Consequences
Visual function of GUCY2D-LCA is severely abnormal.92 All patients have nystagmus
with visual impairment noted in the first year of life. Ophthalmoscopic findings include
retinal vessel attenuation and a granular appearance to the peripheral fundus; macular
pigmentary disturbances have been observed. Visual acuity is abnormal and ranges from
20/80 to no light perception; the level of acuity is not related to age.44,92 Using
acuity as a qualitative severity estimate, 50% of the GUCY2D-LCA patients in our cohort
had CF or worse in the best-seeing eye44 compared with 62% to 89% of CEP290-LCA and
10% of RPE65-LCA. Like CEP290-LCA, the GUCY2D-LCA patients were also hyperopic. Visual
fields by kinetic perimetry can be detectable in some patients, but these are small
central islands and there are occasional peripheral islands with the largest and brightest
target (V-4e). Most patients have nondetectable rod and cone electroretinograms (ERGs)
but up to 25% can show very reduced rod ERG signals.44,92
Cone and rod sensitivities were measurable with FST in almost all patients in our
cohort (Fig. 8). Both photoreceptor-mediated sensitivities showed a range of results
(Fig. 8A). Highest cone sensitivities could be as little as 1 log unit reduced from
mean normal. Unlike in CEP290-LCA, rod sensitivity was detectable in all but a few
patients. By FST, rod sensitivities can be near normal but the range was wide, and
there were also those with minimally or no detectable rod function. Notable in the
cone and rod sensitivities displayed from highest to lowest (Fig. 8A) is the fact
that the patients with the best cone sensitivities are not the same as those with
the highest rod sensitivities. The relationship of rod and cone sensitivity in each
patient can be complex.44
Figure 8
Rod and cone photoreceptor-mediated function in GUCY2D-LCA. Full-field sensitivities
for light-adapted red (red bars) and dark-adapted blue flashes (blue bars) to assess
cone and rod function, respectively. (A) Sensitivities are ranked from high to low
to illustrate the range of dysfunction in GUCY2D-LCA patients. All cone sensitivities
are abnormal by 1 log unit or more, and the range includes very severe dysfunction.
Rod sensitivities could reach normal levels in some patients, and there can be relatively
good rod function in the majority of patients. Note that patient order (horizontal
axis in each graph) is not the same for both rod and cone sensitivity levels, indicating
that patients with better cone function are not necessarily the same as those with
better rod function. (B) Cone and rod function ranked by visual acuity (lowest to
highest, top to bottom). There is a strong relationship between acuity and the cone
metrics, but rod and cone sensitivity are not closely related, as mentioned in (A).
NLP, no light perception; BLP, bare light perception; LP, light perception; HM, hand
motions; CF, count fingers; nd, not detected. Negative-going bars indicate no perception
of the stimuli. Rod sensitivity for patient 21 and patient 24 and cone sensitivity
for patient 24 were very close to instrument limit; these bars were set to 0.1 for
this depiction. Brackets on (A) and bars on the horizontal axes on (B) are centered
on the normal (N) mean sensitivity and the limits represent ±2 SD. Figure courtesy
of Alejandro J. Roman (Scheie Eye Institute, University of Pennsylvania).
Cone vision was related to measurable visual acuity (Fig. 8B); cone sensitivity losses
(CSL) ranged from 1.0 to 4.7 (median 2.8) log units. Rod sensitivity losses (RSL)
ranged from 0.1 to 6.5 (median 1.4) log units. Side-by-side comparison of cone and
rod sensitivity losses shows that many of those patients with low acuity and considerable
cone sensitivity losses could have substantial rod sensitivity by FST and vice versa
(Fig. 8B). We asked whether there was a way to group patients by cone and rod vision
impairment. The answer was that there may be different groups of patients: There were
two small groups and a larger third group that included most of the patients.44 The
two small groups shared severe rod vision disturbances but differed in that one group
had <2 log units of cone vision abnormality and the other had severe cone dysfunction.
The remainder of the patients had less severe rod loss and a spectrum of cone losses,
and the cone and rod sensitivity losses were moderately related.44 Substantial rod
function is thus measurable by full-field psychophysics in GUCY2D-LCA patients; there
was no clear relationship of patient age to presence of residual rod function. The
presence of considerable psychophysically measured rod function was confirmed objectively
in some patients by ERG.92
Photoreceptor Structure
Despite the major losses of photoreceptor function, LCA patients with GUCY2D mutations
present with a mainly intact retina morphologically and there are retained rods and
cones in both the macula and peripheral retina into adulthood.44,92 More specifically,
we first reported that photoreceptor ONL thickness by OCT was within normal limits
except in the foveal region of some patients.92 The foveal region was studied, and
whereas 50% of the patients can have foveal ONL that is within normal limits, almost
all show abnormalities in more distal retinal elements. The foveal bulge (FB) or bowing,
attributed to increased COS length at this locus in normal subjects, is less evident
or not present in most of the GUCY2D-LCA patient scans (Fig. 9A, left). The IS/OS
reflectivity in the patient scans appears thickened and less intense. When we quantified
the FB in normal subjects (n = 10, ages 8–62), the mean FB was 16.08 μm ± 3.18 (mean
± standard deviation, SD). In GUCY2D-LCA patients, mean FB was 4.42 μm ± 5.43. The
difference in FB between the two groups was statistically significant (P ≤ 0.001,
t-test). Only approximately 15% of the patient scans had a FB within 2 SD of the normal
mean.44
Figure 9
Retinal structure at the fovea and perifovea in GUCY2D-LCA. (A) Cross-sectional OCT
scans along the horizontal meridian through the fovea (F) in a normal subject and
seven GUCY2D-LCA patients, ranging in age from 1 to 59 years. Enlarged central scans
(yellow box) in a normal subject and two GUCY2D-LCA patients are also shown. The ONL
is highlighted in blue and the COS layer is in orange. The upper image from a patient
illustrates a thinned COS layer but normal ONL. The lower image is from a patient
with reduced ONL thickness and an interrupted COS layer, suggesting a central absence
of COS. FB, foveal bulge. (B) OCT scans along the vertical meridian including the
fovea and continuing into the superior perifoveal region in a normal subject and four
patients with GUCY2D-LCA ranging in age from 7 to 30 years. Enlarged scans (yellow
box) in a normal subject and a patient showing comparable ONL (blue) and rod outer
segment layer (light blue) thickness. Figure courtesy of Alexander Sumaroka (Scheie
Eye Institute, University of Pennsylvania).
Further measurements of the foveal outer retina suggested a disturbance of COS tips
as they interdigitate with the RPE processes. For example, the magnified and colorized
images of two GUCY2D-LCA patients are compared with the scan of a normal subject (Fig.
9A, right). One patient illustrates slightly reduced foveal ONL and a lack of FB attributable
to COS thinning. The other patient has definitely reduced foveal ONL and a central
region in which the COS layer appears to be lost; this could be an early manifestation
of a hyporeflective zone such as previously described in other maculopathies. COS
thickness was reduced in approximately 85% of patients. In the remainder, the normal
reflective landmarks were not present. We also compared the intensity of the inner
segment/outer segment (IS/OS) band to the intensity of the deeper complex of reflections
composed of COST and RPE to calculate a relative intensity ratio of IS/OS to RPE.
The relative intensity in the group of normal subjects ranged from 0.6 to 0.94, whereas
in GUCY2D-LCA patients with a detectable IS/OS band, the relative intensity ranged
from 0.36 to 0.76. Fifty percent of the patients had a relative intensity that fell
within the narrow interval of 0.5 to 0.6. There was a statistically significant difference
in relative intensity of IS/OS between the control group and GUCY2D-LCA patients.44
The GUCY2D-LCA patients had considerable variation of rod function from nearly normal
to severely abnormal (Fig. 8). Were there any abnormalities of rod photoreceptor retinal
structure? We studied the “rod hotspot” (RHS), the superior retinal region of highest
rod density in the human eye (Fig. 9B). The ONL was within normal limits in all but
one of the patients. When comparing the mean ONL results of normal control subjects
at the RHS and that of GUCY2D-LCA patients, there was no statistically significant
difference between these two groups. There was also no dramatic loss of thickness
of the laminae distal to the ONL; only one patient had rod outer segment (ROS) thickness
that was below the normal limits. A detailed examination of the scans revealed that
the relative intensity of the IS/OS (by comparison with the intensity of the deeper
complex of reflections) was reduced.44
The key issue of structure versus function (and the potential for improvement in vision)
was quantified in the fovea of those GUCY2D-LCA patients with measurable COS length
and definable cone function.44 We asked if the relationship of cone function to cone
structure in GUCY2D-LCA was behaving like a pure photoreceptor degeneration. Loss
of photoreceptor structure is typically the underlying basis for loss of photoreceptor
function. In GUCY2D-LCA patients with measurable foveal ONL and COS, we plotted structure
versus cone function and compared results to those of normal subjects and other IRDs,
specifically RP (Fig. 10A). We defined structure as the product of ONL thickness (a
proxy for photoreceptor numbers) and COS length (a proxy for opsin molecules within
each retained photoreceptor) at the fovea. Cone sensitivity loss was used as a measure
of visual function. We plotted the structure–function data from different forms of
RP and applied a simple linear model that has been used to describe this relationship
in various retinal degenerations. The results indicate that the other RP genotypes
were behaving similarly: Visual sensitivity was reduced linearly with quantum catch.
Most of the GUCY2D-LCA patient data, however, differed from the other genotypes and
fell outside of the 95% confidence interval of normal variability. The current results
support the observation that in most GUCY2D-LCA patients there is a greater degree
of dysfunction than could be explained by the structural loss of cone nuclei and shortening
of COS.
Figure 10
Relation of retinal structure and visual function in GUCY2D-LCA. (A) Relationship
between product of foveal ONL and COS thickness (as a fraction of normal mean) and
visual function (cone sensitivity loss, CSL) in GUCY2D-LCA patients (red), normal
control subjects (gray), and patients with forms of autosomal recessive retinitis
pigmentosa, RP (black). CSL is estimated from FST in GUCY2D-LCA and foveal cone perimetry
in other patients and normal subjects. (B) Relationship between product of rod hotspot
ONL and ROS thickness (as a fraction of normal mean) and visual function (rod sensitivity
loss, RSL) in GUCY2D-LCA patients (blue), normal control subjects (gray), and patients
with various forms of RP (black). RSL is estimated from FST in GUCY2D-LCA and from
dark-adapted perimetry in other patients and normal subjects. The ellipses in (A,
B) enclose the 95% confidence interval of bivariate Gaussian distributions, indicating
the regions of normal variability. Translating the normal variability along an idealized
model for pure photoreceptor degenerations produced a region of uncertainty, which
is shown as the areas bound by the dashed lines. Figure courtesy of Alexander Sumaroka
and Alejandro J. Roman (Scheie Eye Institute, University of Pennsylvania).
As with cone structure and function, we asked if the rod dysfunction in relation to
rod structure in GUCY2D-LCA was behaving like a pure photoreceptor degeneration. All
but one GUCY2D-LCA patient showed a relationship that fell outside of the predicted
model for a pure photoreceptor degeneration (Fig. 10B). There was disproportionate
loss of rod function for the remaining rod structure.
Disease Progression
To date there is no published natural history of the retinal disease in GUCY2D-LCA.
Investigators with quantitative structural and functional data in GUCY2D-LCA patients
should consider performing such a study. A simple route to such data would be to perform
follow-up evaluations on recent examinations (i.e., those with modern LCA-specific
methods) that included potential outcomes for a clinical trial such as OCT (with segmentation
and attention to outer retinal laminae), FST (dark- and light-adapted conditions with
chromatic stimuli to capture rod and cone function), pupillary light responses, and
oculomotor instability (OCI) in addition to conventional techniques such as visual
acuity but with consideration of low-vision methods such as the Berkley Rudimentary
Vision Test.
Postreceptoral Structure Along the Retinocortical Pathway
An appropriate concern to be addressed in pretreatment studies is whether the severe
visual impairments resulting from congenital retinal dysfunction in GUCY2D-LCA may
alter the structure and function of the brain of these individuals and thereby prevent
therapeutic efficacy. We thus studied the postretinal visual pathways in six molecularly
defined GUCY2D-LCA patients using multimodal magnetic resonance imaging (MRI) of the
brain.93 The patients had visual acuities ranging from 20/160 to no light perception,
and all patients had the characteristic findings of severe visual impairment but relatively
preserved retinal structure. As expected, the visual loss in GUCY2D-LCA was accompanied
by attenuation and constriction of the cortical response. Comparisons were made with
normally sighted subjects and other congenitally “blind” subjects with causation that
disrupted retinal structure. GUCY2D-LCA patients showed no difference in size of the
optic chiasm from normal, and this was consistent with our measurements of retinal
GCL thickness by OCT. Blind subjects with other etiologies of disease were significantly
different; that is, the optic chiasm was smaller. Along the visual pathways through
the lateral geniculate nucleus and pericalcarine white matter, the GUCY2D-LCA results
were intermediate between normal and “other blind” subjects but not significantly
different from either group. Like “other blind” subjects, the GUCY2D-LCA patients
had thickened visual cortex gray matter, which is consistent with early and severe
deprivation of form vision.93 Although there remain many questions about the relation
of pretreatment brain measures to posttreatment visual recovery prospects in GUCY2D-LCA,
results to date are sufficiently promising to continue on the path toward treating
the retina of these patients.
Treatment Approaches to GUCY2D-LCA
Preclinical studies in multiple LCA1 murine models have established that using gene
therapy to deliver the GUCY2D gene to the rods and cones of the eye using a recombinant
AAV vector is feasible and efficacious.94
–99 Structural preservation and functional restoration of photoreceptors were achieved
as demonstrated by histologic examinations, ERGs, and improvements in vision-elicited
behavior. Furthermore, AAV-mediated gene replacement has proven safe and effective
for the treatment of other retinal diseases, most notably RPE65-LCA.20
–24,26,29,31
An early phase gene augmentation clinical trial for GUCY2D-LCA should primarily establish
safety of the therapeutic agent (which would be delivered by the subretinal route)
and secondarily determine if there is preliminary evidence of efficacy. A plan would
be to treat a relatively large region of central retina involving not only the cone-rich
fovea but also surrounding rod-rich retina. What parameters could be used to determine
criteria for early and later cohorts and to monitor post treatment for efficacy? Beginning
with functional criteria, it would seem judicious to include in the earlier cohorts
patients with measurable but very reduced visual acuity, and CSL (by FST) between
2 and 4 log units (acuity equivalent of worse than 20/200 and including those with
light perception vision). The considerable range of results for CSL as well as RSL
in GUCY2D-LCA patients leads to the suggestion that earlier cohorts should also have
substantial RSL (again, for example, between 2 and 4 log units). The hypothesis could
then be tested that therapy may affect rod as well as cone function. Later cohorts
could answer questions about the effects of therapy in those patients with relatively
equal CSL but different degrees of RSL and those patients with the most profound CSL
and RSL.
Structural abnormalities showed a gradient in GUCY2D-LCA patients but the gradient
of function is far greater. FB and IS/OS intensity would be valuable to monitor for
changes throughout a trial, but less valuable as criteria for defining cohorts to
enter the trial. Patients with OCT revealing a pattern of severe central photoreceptor
cellular loss could be relegated to later cohorts after it has been determined what
the effects (negative and positive) are of therapy.
CONCLUSIONS
Patients with IRDs seek diagnoses, prognoses, and therapies from their doctors. Most
ophthalmic practitioners are not specialized in such disorders, so the responses to
the many questions posed depend on their previous training about these rare diseases.
The extensive amount of material to learn about treatable ophthalmic diseases makes
IRDs (other than some of the characteristic funduscopic changes) one of the least
emphasized topics during training. What do patients hear from their practitioners
when an ophthalmic examination reveals some pigmentary retinopathy? They are usually
told that their disease is incurable; that it leads to progressive blindness; and
that it can be genetic (implying passed along to further generations). Most depressing
to the affected patient is a frequent remark that further follow-up is not needed,
suggesting that the case is hopeless and of no particular interest to that practitioner.
Times have changed, however, and now doctors can provide more information to IRD patients.
The diagnosis can be defined not only clinically but also by molecular testing. The
genetics in the family can become less of a guess and more certain. A literature of
gene-specific natural history studies is growing, so questions about prognosis may
be able to be answered. And even if the ophthalmic practitioner is not particularly
interested in these rare diseases, patients can be guided to knowledge of their specific
problem by referral to a growing number of IRD specialists. Accurate diagnostics at
the clinical and molecular levels have led to clinical trials for a few of the IRDs.
Given strong preclinical evidence for considering a clinical trial, pharmaceutical
companies are becoming more interested in the concept. The goal of this manuscript
is to point out that careful selection of diseases for treatment in the current era
derives not only from understanding of the gene and the relevant disease models, but
also from decisions about whether human vision can be potentially improved by intervention.
The alternative (and more common) therapies that seek to delay the time course of
visual deterioration need to be preceded by quantitative measures of the disease natural
history. If not, the major effort, expense, and risk of some novel therapies may not
be in the patient's best interest. Advances will continue, and despite the understandable
desperation of a patient for some form of therapy, the ophthalmic practitioner should
be sufficiently educated in relevant progress to provide up-to-date and accurate counsel.