Natural History of Cone Disease in the Murine Model of
Leber Congenital Amaurosis Due to CEP290 Mutation:
Determining the Timing and Expectation of Therapy
Shannon E. Boye1*, Wei-Chieh Huang2, Alejandro J. Roman2, Alexander Sumaroka2, Sanford L. Boye1,
Renee C. Ryals1, Melani B. Olivares2, Qing Ruan1, Budd A. Tucker3, Edwin M. Stone3,4, Anand Swaroop5,
Artur V. Cideciyan2, William W. Hauswirth1, Samuel G. Jacobson2*
1Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, United States of America, 2Scheie Eye Institute, Department of
Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3Stephen A. Wynn Institute for Vision Research, University of Iowa
Carver College of Medicine, Iowa City, Iowa, United States of America, 4Howard Hughes Medical Institute, University of Iowa Carver College of Medicine, Iowa City, Iowa,
United States of America, 5Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
Background: Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause
early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only
mouse model carrying a Cep290 gene mutation, rd16;Nrl2/2, was engineered to mimic the human disease. In the current
study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-
clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and
visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
Methods: Rd16;Nrl2/2mice were studied in the first 90 days of life with optical coherence tomography (OCT),
electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of
patients with CEP290-LCA (n=15; ages 7–48) were compared with those of the model.
Results: CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe
visual loss); the extent of this island declined slowly with age. The rd16;Nrl2/2model also showed a relatively slow
photoreceptor layer decline in thickness with ,80% remaining at 3 months. The number of pseudorosettes also became
reduced. By comparison to single mutant Nrl2/2mice, UV- and M-cone ERGs of rd16;Nrl2/2were at least 1 log unit reduced
at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased
Conclusions: The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both
CEP290-LCA patients and the rd16;Nrl2/2murine model. Pre-clinical proof-of-concept studies for uniocular therapies would
seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular
difference in the UV-cone ERG.
Citation: Boye SE, Huang W-C, Roman AJ, Sumaroka A, Boye SL, et al. (2014) Natural History of Cone Disease in the Murine Model of Leber Congenital Amaurosis
Due to CEP290 Mutation: Determining the Timing and Expectation of Therapy. PLoS ONE 9(3): e92928. doi:10.1371/journal.pone.0092928
Editor: Stephan C.F. Neuhauss, University Zu ¨rich, Switzerland
Received January 26, 2014; Accepted February 26, 2014; Published March 26, 2014
Copyright: ? 2014 Boye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the Grousbeck Foundation, The Foundation Fighting Blindness, Macula Vision Research Foundation, Hope for
Vision, and the NEI intramural research program. AVC is a RPB (Research to Prevent Blindness) Senior Scientific Investigator. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (SGJ); firstname.lastname@example.org (SEB)
Leber congenital amaurosis (LCA) is a molecularly heteroge-
neous group of severe early-onset human diseases [1,2]. By clinical
definition, LCA patients show severe visual loss early in life, but
the morphological basis of the malfunction differs among
genotypes . Visual loss can be due to severe outer retinal
degeneration or retinal disorganization with loss of photoreceptors
in early life. A few molecular forms of LCA, however, have
unexpectedly shown retained outer retinal structure, indicating
structure-function dissociation. For example, the form of LCA
associated with RPE65 (retinal pigment epithelium-specific-65-
kDa) gene mutations can have retained photoreceptors  and
treating regions with residual retinal pigment epithelium (and
photoreceptors) with viral-mediated gene augmentation has led to
remarkable increases in vision. The road to clinical trials of
RPE65-LCA was paved by critical proof-of-concept research with
large and small animal models of the human disease .
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In another molecular form of LCA with severe visual
consequences, CEP290 (cilia-centrosomal protein 290 kDa)-LCA,
patients over a wide age range were shown to retain a central
island of dysfunctional cone photoreceptors [3,6–8]. Early loss of
rod photoreceptors in CEP290-LCA has been documented [6,7].
The survival of the central cones into adulthood makes CEP290-
LCA a worthy target for a macular gene augmentation therapy.
Murine models of the disease recapitulate the rapid rod cell loss of
the human disease [9,10] but the high rod:cone ratio in the mouse
retina complicates proof-of-concept research of cone-selective
therapy. For that reason, a murine model was genetically-
engineered to more closely approximate the human disease. An
all-cone retina with Cep290 mutation, the double mutant
rd16;Nrl2/2, showed dissociation of cone structure and function
(as in the human disease); this determination was made in 3–4-
months-old mice only .
Many questions still need to be answered to enable proof-of
concept studies for therapy to be performed in this model of
human CEP290-LCA? What is the natural history of the cone
disease in this murine model? What would be the optimal timing
of therapeutic intervention in the model? What are the expecta-
tions from such treatment and how long should we wait for a
postulated effect? The present work studies the natural history of
cone structure and function in the rd16;Nrl2/2mouse, confronts
the complexity of the photoreceptor dysmorphogenesis well known
to Nrl2/2mice; determines the window of timing for testing
therapies; and establishes outcomes for determining efficacy. The
results should expedite the pre-clinical steps toward eventual
translation to clinical trials.
Materials and Methods
Human Subjects and Ethics Statement
The human studies were approved by the University of
Pennsylvania Institutional Review Board (IRB; Protocol 226100).
For adult subjects, written informed consent was obtained. For all
children, written parental permission was obtained. Written assent
was obtained from children ages 12 to 17; oral assent was obtained
from children ages 7 to 11; children under the age of 7 years were
enrolled with written parental permission. These consent/assent
procedures were approved by the Institutional review board and
the procedures adhered to the tenets of the Declaration of
Helsinki. The study included 15 patients with LCA due to CEP290
mutations (some phenotypic features of 14 of the patients have
been published) ; the additional patient was a 9-year-old girl
who is a compound heterozygote for IVS26+1655 A.G and
IVS13+1 G.C mutations. There were also 26 patients with
retinal degeneration including simplex/multiplex retinitis pigmen-
tosa (RP), X-linked RP and Usher syndrome. Patients underwent a
complete eye examination, including visual acuity and optical
coherence tomography (OCT). Data will be made freely available
Retinal cross-sectional imaging with 9-mm (30-degree) line
scans along the horizontal meridian crossing the fovea was mainly
performed with a spectral-domain OCT unit (RTVue-100;
Optovue, Fremont, CA); images from a subset of patients were
acquired using a time-domain OCT unit (OCT3; Carl Zeiss
Meditec, Dublin, CA). Two novel parameters of foveal structure
were derived from CEP290-LCA OCT data that were mostly (14
of 15) collected during previous studies [3,6,7]. The first parameter
was average ONL thickness of the foveal region which was defined
as the average of 5 samples obtained at the foveal pit and two on
each side of the fovea along the vertical meridian at 0.15 and
0.3 mm eccentricity. The second parameter was the width of the
foveal ONL island which was defined as the retinal distance
between two points at half maximum ONL.
Animals and Ethics Statement
All experiments were performed in strict accordance with the
recommendations in the Guide for Care and Use of Laboratory
Animals of the National Institutes of Health and the USDA’s
Animal Welfare Act and Animal Welfare Regulations, and
complied with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. The protocols were approved
by the Institutional Animal Care and Use Committee of the
University of Florida (IACUC Protocol # 201304739) and the
University of Pennsylvania (IACUC Protocol #804387). Data will
be made freely available upon request. The rd16;Nrl2/2double
mutant mice and C57BL/6 control mice (Jackson Laboratory, Bar
Harbor, ME) were maintained in the University of Florida Health
Science Center’s animal care facilities in controlled ambient
illumination on a 12-hour light/12-hour dark cycle (ambient
illumination, ,3 lux). Access to food and water was ad libitum.
ERGs were performed with published methods [7,11,12] using
general anesthesia (ketamine HCl, 65 mg/kg, xylazine, 5 mg/kg,
intraperitoneal injection) and topical corneal anesthetic (propar-
acaine HCl). Pupils were dilated (tropicamide 1%, phenylephrine
2.5%). A computer-based system (Espion, Diagnosys LLC,
(40 cd.m22white) ERGs in response to a Xenon UV flash
(360 nm peak, Hoya U-360 filter, Edmund Optics, Barrington,
NJ, USA) and to a green flash produced with LEDs (510 nm peak;
0.87 log phot-cd.s.m22, 4 ms). The energy of the UV flashes was
adjusted to evoke responses matched in waveform to those elicited
by the green stimulus in WT mice. The stimuli were presented in a
ganzfeld lined with aluminum foil [7,11,12].
to record light-adapted
Optical Coherence Tomography
Retinal OCT imaging was performed as previously described
[12,13]. Retinal cross-sectional images of rd16;Nrl2/2and control
mice were acquired with a spectral domain (SD) OCT system
(Bioptigen, Inc., Durham, NC). Animals were anesthetized and
pupils were dilated as for ERG recordings. Corneas were
lubricated during the imaging session (Systane Ultra ophthalmic
lubricant; Alcon Ltd., Fort Worth, TX). The optic nerve head
(ONH) was centered within a 1.5 mm diameter field of view under
fast fundus mode, using a raster consisting of 200 parallel b-scans
of 200 longitudinal reflectivity profiles (LRPs) per scan line .
High-resolution scans (40 parallel b-scans of 1000 LRPs in each
scan line, each repeated four times) were acquired along the
horizontal (nasal-temporal) and vertical (dorsal-ventral; superior-
inferior) axes. Repositioning of the eyes then occurred and the
ONH was placed at top or bottom center of the view. High-
resolution scans were repeated at these locations for coverage up to
Post-acquisition processing of OCT data was performed with
custom programs (MATLAB 7.5; MathWorks, Natick, MA) and
commercial software (InVivoVue Clinic software; Bioptigen, Inc.).
Four repetitions of the high-resolution scans were averaged using
the manufacturer’s software. Vertical scans with ONH at the
center, and those superior and inferior to the center of the ONH
were montaged by custom programs. LRPs of the merged OCT
images were aligned by manually straightening the retinal pigment
epithelium (RPE) reflection , which was defined as the second
hyperreflective band from the sclerad side . Retinal thickness is
the distance from the vitreoretinal interface and the RPE peak.
CEP290 LCA Mouse
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Photoreceptor (termed ONL+) thickness is defined as the distance
between the signal trough delimited by the signal peak defining the
sclerad side of the outer plexiform layer to the signal trough vitreal
to the hyperreflective band of the RPE. When data are
represented as profiles across the vertical meridian, the average
and SD values are calculated from all data points in the vertical
profile excluding 0.18 mm around ONH. Photoreceptor ONL+
and ONL fractions, OCT and histology measurements respec-
tively, were estimated by normalizing to the mean values of the
youngest age studied (P31 for OCT, n=10 eyes, and P21–40 for
histology, n=6 eyes, measurements).
Retinal en face reflectance images to enable counts of pseudoro-
settes were generated using 350 by 350 horizontal raster scans
centered within the 1.5 mm diameter field of view. Spatial
distribution of pseudorosettes around the ONH was revealed by
integrating the OCT backscatter intensity between two boundaries
that envelope all rosettes in the OCT raster images. The light-
scattering pseudorosettes appear as white spots in the en face image.
Total numbers of pseudorosettes in the sampled area were counted
manually using ImageJ (http://rsb.info.nih.gov/ij/). Comparisons
between groups were performed using unpaired, two-tailed t-tests.
Significance is defined as a p value ,0.05. Density of pseudoro-
settes in different sectors within the central retinal region was also
quantified (n=8 eyes for two age groups).
Histopathology and Immunocytochemistry
Eyes of rd16;Nrl2/2mice were enucleated at postnatal day 21
(P21), P40, P60 and P80 and immediately placed in 4%
paraformaldehyde for 24 hours. P60 WT mice were used as
controls. Following incubation in 30% sucrose/1X PBS (2 hrs),
tissue was prepared for cryoprotection and sectioning as previously
described . Ten micron sections were cut on a cryostat (Leica
CM3050 S, Wetzlar, Germany). Sections designated for H&E
staining were rehydrated for 10 minutes, followed by immersion in
95% EtOH for 2 minutes, 70% EtOH for 2 minutes and a wash
with distilled H2O. Harris’s hematoxylin solution (RICCA Cat.
No. 3530-1, Arlington, TX) was applied for 6 minutes followed by
a 1 minute wash in running tap water. 0.2% ammonia ‘‘bluing’’
solution (Fisher chemicals #A669-500, Waltham, MA 02454) was
applied for 45 seconds, followed by a wash in running tap water.
This was followed with ten successive immersions in 95% EtOH
and a counterstain in eosin Y solution (RICCA Cat. No. 2850-16,
Arlington, TX) for 6 minutes. Sections were then dehydrated with
95% EtOH and 2 immersions in absolute EtOH for 5 minutes
each. Following immersion in deionized H2O, AQUA-MOUNT
mounting media was applied and slides were coverslipped.
Sections designed for immunohistochemistry were processed as
follows. After rinsing with 1X PBS, sections were incubated with
0.5% Triton X2100 for 1 hour followed by a 30 min incubation
with a blocking solution of 1% bovine serum albumin (BSA).
Retinal sections were then incubated overnight at 4uC with rabbit
polyclonal antibodies raised against cone transducin alpha
(GNAT2) (sc-390, Santa Cruz, 1:500) or S cone opsin (AB5407,
Chemicon International, 1:300) and lectin PNA conjugated to
Alexa Fluor 594 (L32459, Invitrogen, 1:400) diluted in 0.3%
Triton X2100/1% BSA. The following day, sections were rinsed
with 1X PBS and incubated for one hour at room temperature in
anti-rabbit IgG secondary antibody Alexa-fluor 488 (Invitrogen,
Eugene, Oregon, Cat#A11008) diluted in 1X PBS at 1:500. After
a wash, sections were cover slipped with SlowFade Gold antifade
reagent with DAPI (S36939, Life Technologies). Retinal sections
were imaged at 5X using a fluorescent Axiophot microscope
(Zeiss, Thornwood, NY). 20X images were captured with spinning
disk confocal microscopy (Nikon Eclipse TE2000 microscope
equipped with Perkin Elmer Ultraview Modular Laser System and
Hamamatsu O-RCA-R2 camera). Exposure settings were consis-
tent across images at each magnification.
Pseudorosettes were counted in sections from cuts through the
globe representing the far peripheral retina in three rd16;Nrl2/2
mice each from the different age groups; comparison with any
regional differences from OCT was not possible because attention
to exact dorsal-ventral orientation of the tissue did not occur. ONL
thickness was measured using Volocity software (Perkin Elmer) in
two peripheral retinal cuts from three rd16;Nrl2/2animals. Counts
were made in four equally-spaced quadrants in each far peripheral
section. Peripheral ONL thickness measurements and pseudoro-
sette counts were averaged and compared across ages using
unpaired, two-tailed t-tests. Significance is defined as a p value ,
Structure and Function in Human CEP290-LCA
LCA caused by CEP290 mutations can show a wide spectrum of
visual acuity results but, in general, most of the measurable values
are severely reduced [16,17]. It was thus surprising when we
observed by OCT retinal imaging that there were foveal islands of
retained photoreceptor nuclei in nearly all such patients [3,6,7]. A
cross-sectional image horizontally across the retina through the
fovea of a 23-year-old normal subject with logMAR of 0 (Snellen,
20/20) shows a continuous photoreceptor outer nuclear layer
(ONL) with adjacent laminar architecture representing synaptic
and nuclear layers to the vitreal side, and inner and outer segment
and RPE layers to the choroidal side (Fig. 1A, upper panel). In
contrast, a 9-year-old LCA patient with no light perception (NLP)
and mutant CEP290 alleles retains a central island of ONL in the
cone-rich fovea that is within normal limits for thickness, although
the inner and outer segment laminar architecture is abnormal
(Fig. 1A, middle panel) . There is no measurable ONL beyond
the central island. An RP patient (age 17) shows a similar central
island of ONL, but has a visual acuity of 0.3 (20/40; Fig. 1A, lower
panel); the distal laminar architecture representing outer and inner
segments approximates that of the normal subject in the very
To enable comparisons to be made between human CEP290-
LCA function and structure and that of the rd16;Nrl2/2mouse
model, we plotted the relationship between visual acuity and foveal
ONL thickness in a cohort of CEP290-LCA patients (n=19 eyes of
15 patients, ages 7–48 years; Fig. 1B) . Eleven of the 19 eyes
had a foveal ONL thickness of .79.3 mm, which represents the
lower limit of normal (mean 6 SD, 93.767.2 mm; n=10). There
was a wide range of abnormal visual acuities (from 0.4 logMAR to
NLP) but 11/19 eyes (57%) of the 15 patients in this cohort have
HM (hand motions) vision or worse. Linear regression through
these CEP290-LCA patient data had a slope of 20.02 (r2, 0.027)
and this was not significantly different from 0 (p=0.49). In other
words, there was no decrease in foveal ONL thickness associated
with lower acuities. This is in contrast to the data from a group of
patients with other retinal degenerative diseases, collectively
termed RP (n=26 patients; ages 10–69; Fig. 1B, inset). Unlike
in CEP290-LCA, the RP patients show the more expected
relationship: reduction in visual acuity is accompanied by
decreasing foveal ONL thickness [4,18].
Does the island of cone photoreceptor nuclei remain stable with
age in CEP290-LCA? Without longitudinal data available in our
cohort, we used cross-sectional data to quantify the width of the
central island along the horizontal meridian in 14 patients
(Fig. 1C). The width of the central island tends to decrease with
CEP290 LCA Mouse
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age (slope of 21.96; r2, 0.19), suggesting that a slow degenerative
process is likely ongoing in these patients, as it is in all retinal
Retinal Structure in rd16;Nrl2/2mice at Different Ages
Optical imaging provided the opportunity to study the retinal
morphological changes at different ages in the rd16;Nrl2/2mice.
Representative vertical OCT scans across the ONH are shown
from a wild-type (WT) mouse at age 90 days (P90), and rd16;Nrl2/
2mice at P31 and P83 (Fig. 2A). The scans from the mutant mice
differ from that of the WT mouse. For example, total retinal
thickness differs as well as the thickness of the photoreceptor
layers. WT mice, of course, have a different complement of
photoreceptors than the rd16;Nrl2/2mice. WT have mainly rods
and a minority of cones, while the mutant mice have exclusively
cone photoreceptors [7,11,19]. The mutant mice also show
hyperreflective loci (presumed pseudorosettes) within the outer
retinal layers and extending into the inner retina; the loci are most
evident in the inferior retinal part of the section. The hyperre-
flectivities appear more prominent in P31 than in P83. Magnified
sections from the three scans indicate some of the difficulty in
defining the outer retinal layers in the rd16;Nrl2/2mice compared
with WT. ONL and the laminae attributed to IS and OS (distal to
the OLM) are readily discernible in WT cross-sectional images, as
is the RPE hyperreflection [12,13]. The rd16;Nrl2/2mice present
a different pattern of reflectivities with an indistinct OLM and thus
no clear delineation of the laminar architecture of the IS and OS
of these cone cells (scattering band distal to ONL is labeled S+) .
An RPE hyperreflectivity, however, is discernible (Fig. 2A lower
panels). ONL thickness appears reduced in P83 compared to P31.
Photoreceptor layer thickness across the ONH along the vertical
meridian (central 2.4 mm of retina) is quantified for two age
groups of mutant mice, ages 31–41 and 83–89 days (Fig. 2B). The
profiles of photoreceptor laminae at P31–41 appeared spiky
because of the disturbances in the outer layers by the pseudoro-
sette structures (Fig. 2A; Fig. 2B, inset near P31–41 panel). The
profiles, when areas with pseudorosettes were specifically exclud-
ed, however, become smoother as would be expected for a
photoreceptor layer . The spiky appearance in the vertical
profiles diminishes in the older age group, suggesting a reduction
in pseudorosettes with age in the mutants (Fig. 2B, inset near P83–
89 panel). The photoreceptor laminae across the vertical meridian
in the P31–41 age group is significantly thicker than in the older
age group (mean 6 SD, P31–41: 66.467.6 mm; P83–89:
54.767.0 mm; p,0.001), indicating a decline with age in the
mutants. When the spiky profile data that included the pseudoro-
settes was similarly analyzed, there was also a significant difference
between ages (P31–41: 69.469.7 mm; P83–89: 54.467.6 mm; p,
0.001). There is an apparent superior-inferior (dorsal-ventral)
asymmetry with the P83–89 age group showing greater thinning in
the inferior region than superiorly (Fig. 2B). We explored this
possible regional variation. First, we compared the inferior and
superior photoreceptor layer thicknesses at the P31–41 age group
66.864.9 mm; versus Inf: 6766.3 mm; p=0.85). Then we did
the same comparison for the older age group; there was a
difference with inferior retina being thinner than superior retina
(P83–89 Sup: 57.965.5 mm; versus Inf: 51.663.4 mm; p=0.006).
Figure 1. Structure and function in the central retina of CEP290-
LCA patients. (A) Cross-sectional OCT scans along the horizontal
meridian through the fovea in a normal subject, a CEP290-LCA patient,
and an RP patient. ONL is highlighted in blue. Inset shows location of
scan. (B) Relationship of foveal ONL thickness and visual acuity in
CEP290-LCA patients. Bar graph represents the average 61SD foveal
ONL thickness of eyes in the different visual acuity ranges (n=3, for 0.1–
1 LogMAR; n=5, for 1–2 LogMAR; and n=11, for 2–NLP). Dashed line is
lower limit of normal and emphasizes that despite low acuities, foveal
ONL is within normal limits. Inset, data from a series of RP patients
plotted similarly to show the more expected relationship between
structure and visual acuity in retinal degenerations (n=3, for 0–0.2
LogMAR; n=20, for 0.2–1 LogMAR; and n=3 for 1–2 LogMAR). Dashed
line is also lower limit of normal. (C) Relationship in CEP290-LCA patients
of width of the ONL in the central retina and patient age at time of
examination. ONL width was unable to be defined in a 32-year-old
CEP290-LCA patient with maculopathy. Solid line is linear regression.
Inset, traced central ONL peaks in representative patients of different
CEP290 LCA Mouse
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Figure 2. OCT abnormalities in rd16;Nrl2/2mice. (A) Upper panels: Representative OCT scans vertically across ,2 mm of retina (centered at the
ONH, optic nerve head) in a WT mouse and in two rd16;Nrl2/2mice of different ages. Lower panels: Magnified parts of the superior region of the
retinal sections with overlaid longitudinal reflectivity profiles (LRPs) to demonstrate the reflective abnormalities in the outer retinal region in
rd16;Nrl2/2mice (b and c) compared with C57BL6 WT (a). (B) Upper two panels: Vertical OCT sections quantified for ONL+ thickness in two age
groups of rd16;Nrl2/2mice. Regions of outer retina with pseudorosettes were excluded in the measurement. ONL+ profiles in the older (P83–89,
n=12 eyes) age group were thinner than those in younger (P31–41, n=35 eyes) mice; gray bands in the P83–89 plot represent mean62 SD for ONL+
thickness of the P31–41 mice. For reference, insets at lower right of the upper two plots show original raw data before suppression of pseudorosette
regions. Third panel from top: Means of ONL+ data across the vertical meridian in two age groups (error bar: 6 SD; P31–41, open circles; P83–89, filled
triangles). Lowest panel: Histograms showing average ONL+ fraction across vertical meridian of two age groups (*represents p,0.001). (C)
Histological sections of rd16;Nrl2/2retina at 4 different ages from P21 to P80, compared with a WT retinal section. Histograms show ONL fraction
(based on the earlier age group) in rd16;Nrl2/2mice from peripheral retina (n=6 eyes in each of the two age groups, *represents p=0.01).
CEP290 LCA Mouse
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Histograms are shown of the photoreceptor laminar thickness
(average across the vertical meridian) for the two age groups of
mutant mice, expressed as a fraction of the earlier timepoint
(Fig. 2B, lowest panel). Total retinal thickness was also measured
in the scans from the two age groups and these results were
consistent with those of the photoreceptor laminae; there was a
significant difference between ages with older group thinner than
the younger (P31–41: 197.4613 mm; P83–89: 165.5634.4 mm;
p,0.001). Considering that these non-invasive imaging data
represent the more central retina of the mouse eye, we also
measured ONL thickness in histological sections from the
peripheral retina (Fig. 2C). Histological sections of mutant mice
at different ages also illustrate the differences in laminar
architecture compared to WT. Rd16;Nrl2/2show pseudorosettes,
limited IS/OS material and apparent retinal thinning over this
time period (Fig. 2C). Two timepoints similar to those from the
in vivo imaging studies were chosen and histograms of ONL
thickness were plotted as a fraction of the earlier timepoint. In
summary, there is a detectably thinner photoreceptor layer
thickness at the older age using both methods and in different
retinal regions (Fig. 2C).
Pseudorosettes: Topographical Distribution and Age
Histological sections from the peripheral retina in two
rd16;Nrl2/2mice of different ages indicate the presence of
pseudorosettes at both earlier and later timepoints (Fig. 3A). In
the central retina (1.5 mm diameter) we quantified the number
and location of pseudorosettes using en face imaging (Fig. 3B).
Integration of backscatter intensity between boundaries envelop-
ing all pseudorosettes in the raster images produced fundus images
with white spots that represent the pseudorosettes [19–22}
(Fig. 3B). Localization of individual pseudorosettes was then
recorded with respect to the ONH. Pseudorosettes are detected in
all quadrants of the retina in both younger (exemplified by a P31
eye) and older (exemplified by a P83 eye) rd16;Nrl2/2mice
(Fig. 3C). In the older mutant mice, pseudorosettes appeared less
dense but similar in pattern to the younger mutants. There was
also an apparent regional variation with superior (dorsal) retina
having a lesser number of these structures compared to the inferior
(ventral) retina. A comparison of superior versus inferior
pseudorosette counts in both age groups showed that the
superior-inferior difference was significant (P31: n=8 eyes,
average number of pseudorosettes 6 SD; Sup: 44611 versus
Inf: 73612, p,0.001; P83: n=8 eyes, Sup: 24611 versus Inf:
44613, p,0.001). These regional variations of the structures
prompted us to examine the average density in different sectors
within the central retina (Fig. 3C insets, n=8 eyes for both age
groups). Pseudorosette density was found to be higher in the
inferior and temporal sectors than in the superior and nasal retina.
Two further comparisons were made – one by OCT in the
central retina and the other by histology in the peripheral retina
(Fig. 3D). The number of pseudorosettes in the central area
sampled was compared statistically in the two age groups (Fig. 3D,
upper histograms) and there was a significantly lower number in
the older versus the younger mutants (p,0.001). Number of
pseudorosettes were also counted in histological sections from the
peripheral retina of the mutant mice and there was a significant
difference between P21–40 and the P60–80 groups (p=0.01) with,
again, a reduced number of these structures at older ages (Fig. 3D,
Expression of Phototransduction Proteins in rd16;Nrl2/2
mice at Different Ages
Previous studies have shown that loss of full length CEP290
expression leads to mislocalization and/or loss of photoreceptor
proteins [7,9,23]. Here we evaluate whether this phenomenon is
also observed on an Nrl2/2background. We show that expression
of cone phototransduction proteins including the alpha subunit of
cone transducin (GNAT2) and S-cone opsin decrease over time in
the rd16;Nrl2/2mice. Loss of expression was evident in both
central and peripheral retina (Fig. 4A,B). Regardless of age, both
GNAT2 and S-opsin localized to remnant IS/OS of rd16;Nrl2/2
photoreceptors. The most pronounced loss of both proteins
appeared to occur between P40–P60, with very little of each
detected via immunohistochemistry at P80. Despite loss of
phototransduction proteins, cone outer segment sheaths, as
detected by conjugation with peanut agglutinin (PNA), remained
intact at all ages studied (Fig. 4A,B). This suggests that loss of
phototransduction proteins in rd16;Nrl2/2is the result of their
downregulation and/or increased turnover rather than loss of
outer segments or photoreceptor degeneration.
Cone Function in rd16;Nrl2/2mice and Relationship to
ERG responses elicited with UV- and M-cone stimuli in
rd16;Nl2/2mice over the age range from ,30 to ,90 days of age
are illustrated and b-wave amplitudes are plotted (Fig. 5A).
Responses from a limited number of Nrl2/2[11,19] and rd16 
mice are shown for comparison. UV-cone responses in the Nrl2/2
mice are ,1 log-unit higher in amplitude than those in rd16;Nrl2/
2at the younger ages depicted. The Nrl2/2responses show no
change over the time period studied. In contrast, the rd16 mice
have responses that are almost reaching noise levels by day 44, and
are ,1 log unit lower in amplitude than the rd16;Nrl2/2at that
age. Between these two extremes are the data from the rd16;Nrl2/
2mice, which show a measurable decline from an average of
139 mV (range, 103–210 mV) at 31 days to 22 mV (range, 13–
36 mV) by age 83 days. The natural history of function loss is
consistent with an underlying exponential decay which shows
linear reduction of amplitudes on log-linear plots (Fig. 5A).
M-cone ERGs in the rd16;Nrl2/2mice are substantially lower in
amplitude than the UV-cone ERGs. The Nrl2/2mice show
responses about 1 log unit higher in amplitude than those of the
double mutants at the earlier ages and there is no reduction in
amplitude over the ages sampled. Limited data from rd16 mice
were recordable before 44 days of age and within the range of
response amplitudes of the double mutants. In rd16;Nrl2/2mice
there is a measurable decline of M-cone ERG amplitude with age
The structure-function relationship we plotted for human
CEP290-LCA (Fig. 1B) can now be compared to a structure-
function relationship in the rd16;Nrl2/2mouse model (Fig. 5B).
Despite very different types of data and measurements, we tried to
plot the human results for structure (by OCT) and function (by
visual acuity) and the mouse data for structure (OCT) and function
(by ERG amplitudes) in a way that allowed some comparison,
even if by simple observation. Photoreceptor layer (ONL+)
thickness and cone ERG amplitudes (using the sum of UV- and
M-cone ERG amplitudes) were normalized to the earliest
timepoint studied (P31). Cone ERGs in the double mutant,
compared with the all-cone Nrl2/2mice without the Cep290
mutation, are quite reduced in amplitude and, like the visual
acuities of CEP290-LCA, show a range of abnormalities that only
worsens over the age period studied (Fig. 5A). Cone photoreceptor
CEP290 LCA Mouse
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Figure 3. Spatial and temporal distribution of pseudorosettes in rd16;Nrl2/2retina. (A) Histological sections from peripheral retina of two
rd16;Nrl2/2mice at different ages demonstrating the presence of pseudorosettes (arrows). Calibration=50 mm. (B) Schematic drawing of the mouse
CEP290 LCA Mouse
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structure shows change but it is relatively limited, only becoming
evident at the lowest ERG amplitudes (Fig. 5B).
Developing a Strategy for Proof-of-concept Studies
What did we learn from the studies of the rd16;Nrl2/2model of
CEP290-LCA that would guide strategy for proof-of-concept
research using, for example, gene augmentation therapy by
subretinal delivery? The relatively persistent cone photoreceptor
cell layer thickness over the first three months of life and the
measurable ERG responses during these ages provides an
opportunity for intervention. What are the factors that constrain
the timing of intervention and of reassessment to determine
efficacy or toxicity? The slow loss of ONL thickness during this
interval would not be limiting. The declining ERG amplitudes, the
loss of phototransduction proteins, and the onset time for
transgene expression of the viral vector, however, would dictate
the timing of intervention.
The ERG response to assay, given the major difference between
UV- and M-cone amplitudes, is the UV-cone b-wave which
declined at a rate of about 4% per day over the period from 1 to 3
months of age. The basis for the loss of cone amplitude was not
able to be determined in this study, but likely contributors could be
changes in the length of the already diminutive outer segments and
diminishing phototransduction proteins therein. Our data suggest
that a uniocular subretinal injection of vector-gene occur at age
P35–40, when UV-cone ERG responses are still substantial: mean
84 mV (1.960.4 log mV, 95% prediction interval). Four weeks
thereafter, at P65, there would be functional assessment with
bilateral ERGs. At this age, responses in the untreated eye would
be expected to be lower but clearly distinguishable from noise
levels: mean 25 mV (1.460.4 log mV). Interocular asymmetries
(60.3 log)  could then be used to assess treatment efficacy (.
50 mV) or toxicity (,12 mV). Such a strategy of interocular
difference assessment by ERG has been used in prior murine
studies of therapeutic efficacy . Of course, an earlier age for
retina indicating the coverage of the central OCT raster scans (red circle). ONH is centered in the drawing. Integrated en face image of the central
region of a P41 rd16;Nrl2/2mouse showing how pseudorosettes appear as white dots (B, right panel, red circle). (C) Pseudorosette distribution within
the central retinal region in a young (P31) and an older (P83) rd16;Nrl2/2eyes. Insets (up and right) show average pseudorosettes as density in
different sectors of the central retinal region sampled (n=8 eyes for both age groups). (D) Upper: Histograms comparing number of pseudorosettes
in the central retina by OCT at two different ages (P31, n=10 eyes; P83, n=8 eyes). Lower: Pseudorosette counts from histological sections of
peripheral retina of two different age groups (P21–40, n=6 eyes; P60–80, n=6 eyes). Both data sets in rd16;Nrl2/2mice indicate that the number of
rosettes decreases with age (*represents p,0.001 and p=0.01 for the upper and lower graphs, respectively). Error bars, 6 SD from the mean.
Figure 4. Expression of photoreceptor proteins is reduced over time in rd16;Nrl2/2mice. Representative cross sections (20X) from central
and peripheral retina of P21, P40, P60 and P80 rd16;Nrl2/2mice were immunostained for the presence of cone transducin alpha (GNAT2) (A) or S-
cone opsin (B) and PNA (A,B). Despite maintenance of cone outer segment sheaths (PNA), both GNAT2 and S-opsin expression are markedly reduced
by P60 in both central and peripheral retina. INL- inner nuclear layer, ONL- outer nuclear layer, IS/OS- inner segments/outer segments.
CEP290 LCA Mouse
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Figure 5. Structure and function in the rd16;Nrl2/2mouse retina. (A) ERG b-wave amplitudes of responses to UV- and M-cone stimuli as a
function of age in rd16;Nrl2/2mice from P34 to P83 (n=95) with comparisons to data from previously recorded signals in Nrl2/2(squares ; square
with cross ), and rd16 (crosses ) mice. Upper: Cone b-wave responses to ultraviolet (UV, 360 nm peak) stimuli in the rd16;Nrl2/2mice are
severely reduced compared with those of Nrl2/2mice at comparable ages. Amplitudes in rd16 mice are low compared to the other mice. It is also
notable that ERGs of the Nrl2/2mice remain relatively stable throughout this age range, while ERGs of the rd16; Nrl2/2and rd16 mice decline in
amplitude with age. Lower: Responses to green (510 nm) stimuli are substantially lower in amplitude than those from UV-cone stimuli. Again, Nrl2/2
mice have the largest amplitudes and do not decline with increasing age within this time period. The rd16;Nrl2/2waveforms are lower in amplitude
CEP290 LCA Mouse
PLOS ONE | www.plosone.org9 March 2014 | Volume 9 | Issue 3 | e92928
initiation of therapy is also possible but we have no data to support
What are other uncertainties in this theoretical experiment?
Subretinal injections and resulting retinal detachments are well-
known to have negative effects on photoreceptor outer segments as
a result of trauma [25,26]. It has not been established from earlier
work how fragile the shortened outer segments are in either Nrl2/
2mice or rd16;Nrl2/2double mutants. Nrl2/2mice have been
used to engineer cone-only mouse mutants with other genetic
abnormalities [27–29]. It is promising that GFP expression has
been obtained in Nrl2/2mice with subretinal AAV , and
preliminary results suggest efficacy after subretinal gene therapy in
an Nrl2/2;Cnga32/2double mutant .
Another issue to be addressed is the dysplastic phenotype, i.e.
the presence of pseudorosettes, that we quantified in this study.
From the present results, the superior retina showed less numbers
of pseudorosettes at the early and later ages sampled. The finding
of less degeneration in the superior retina would be consistent with
results of previous studies suggesting a relationship of the dysplastic
phenotype and eventual degeneration in mouse mutants including
Nrl2/2mice [12,19,20,22]. If technically feasible to place subretinal
injections mainly in the superior retina, it should avoid the greater
complexity of the inferior retina where we showed a higher density
of pseudorosettes. Targeting treatment to specific retinal regions in
humans undergoing subretinal gene therapy has been valuable in
the RPE65-LCA trials ; in this rd16;Nrl2/2mouse mutant, it
would seem worthy to attempt, if surgically possible.
Viral Vector Considerations for Eventual Delivery of
Cep290 to Cone Photoreceptors
Which vector platform will be useful for proof-of-concept gene
replacement studies in rd16;Nrl2/2mice? Choice must be dictated
at least by the ability of vector to transduce the target cell
(photoreceptors) and mediate persistent transgene expression. If
delivery of full length Cep290 is necessary (cDNA=,7.4 kb),
vector must also accommodate a relatively large genetic payload.
Lentivirus can accommodate full length Cep290 cDNA. Equine
infections anemia virus (EIAV)-based lentivirus, used in concert
with various photoreceptor-specific promoters, showed modest
expression of reporter gene in photoreceptors of mice subretinally
injected at postnatal day 5 (P5), a timepoint prior to photoreceptor
differentiation . EIAV containing photoreceptor-specific
ABCA4 (another relatively large cDNA) restored the disease
phenotype of Abca42/2mice following subretinal injection at
P4–P5 . Transduction of postmitotic photoreceptors has been
more challenging in rodents [34–39] but not in primates .
Adeno-associated virus (AAV) has proven useful for driving
persistent therapeutic transgene expression in postmitotic photo-
receptors [41–44] and has demonstrated safety and efficacy in
proof-of-concept studies  as well as clinical trials [46–49]. The
Cep290 cDNA exceeds the packaging capacity of conventional
AAV (,5 kb). Recently, however, it was reported that AAV is
capable of delivering large cDNAs. Several groups demonstrated
that large cDNAs are randomly truncated during packaging [50–
53]. Upon co-infection, fragmented genomes most likely undergo
homologous recombination leading ultimately to expression of full
length protein. Fragmented AAV (‘‘fAAV’’) has been used
successfully to correct the retinal phenotypes of multiple mouse
models of inherited retinal disease [54–56]. Because the hetero-
geneous nature of genetic payloads in ‘‘fAAV’’ vectors may limit
their therapeutic application, various dual AAV vector platforms
in which single, defined DNA species are encapsidated (vector 1
containing the 59 portion of the cDNA and vector 2 containing the
39 portion of the cDNA) have been developed. Such platforms
have proven useful for delivery of a variety of large cDNAs [57–
What if the entire CEP290 protein (2479 amino acids) was not
required for treatment? Recently, a zebrafish model of CEP290-
LCA was created using morpholinos to generate an altered cep290
splice product that creates a premature stop codon at amino acid
988, modeling the most common CEP290-LCA mutation .
Mutants displayed reduced visual function and delays in melano-
some transport. Embryonic delivery of the N’- terminal portion
alone (the first 1059 amino acids) was sufficient to restore the
vision defect in this model . A C’ terminal fragment (a.a.
1765–2479) proved useful only for restoring melanosome trans-
port. Unlike the N’ terminal mutation in zebrafish, the rd16 mouse
carries a mutation close to the C’ terminus of Cep290 (in frame
deletion of amino acids 1599–1897) , suggesting that this is the
region of the protein required for vision. Reasons for this
discrepancy have yet to be elucidated. It is possible that the rd16
deletion mutation alters Cep290 folding, preventing the N’
terminus from performing an essential function.
It is known that the C’ and N’ termini of CEP290 can interact
with themselves and one another . A recent report shows that
CEP290 activity is auto-inhibited via interactions between its N’
and C’ termini . Full length CEP290 exhibited attenuated
activity in hTERT-RPE1 cells whereas overexpression of N’ or C’
terminal portions alone resulted in aberrant primary cilium
formation. Taken together, these studies suggest that the N’ and
C’ termini physically interact to inhibit CEP290 function.
Overexpression of N’ or C’ terminus alone in this cell line (which
expresses endogenous full length protein), likely led to saturation of
full length CEP290 regulatory domains and a resulting increase in
protein function. It was suggested that truncation mutants of
CEP290 that lack inhibitory domains but maintain other
functional regions of the protein may prove useful (and perhaps
even fit inside a conventional AAV vector) for the treatment of this
disease. Future proof-of-concept studies with lentivirus, ‘‘fAAV’’,
dual AAV vector platforms carrying full length CEP290, and
standard AAV carrying CEP290 truncation mutants are certainly
warranted. The current work defines a specific strategy to evaluate
the ability of such vectors to restore the retinal phenotype of the
Conceived and designed the experiments: SGJ SEB AVC A. Swaroop
EMS WWH. Performed the experiments: WCH AJR A. Sumaroka SLB
RCR MBO QR BAT. Analyzed the data: WCH AJR A. Sumaroka SLB
RCR MBO QR BAT. Wrote the paper: SGJ SEB AVC.
and there is a reduction with age. Only limited data were available for rd16 mice and these fell within the range of rd16;Nrl2/2amplitudes. Waveforms
for representative rd16;Nrl2/2mice at various ages (grey-filled circles) are illustrated in the panels at right. Grey lines: linear regression fit to log-
converted data (dashes) and 95% prediction intervals (solid). Squares with cross at earliest age in graphs: Nrl2/2data from Mears et al., 2001 (B)
Photoreceptor structure (ONL+) as a function of the combined UV- and M-cone ERG b-wave amplitudes. ONL+ remains similar to the value at P31
(youngest age rd16;Nrl2/2we studied) across various degrees of ERG amplitude reduction. Horizontal dashed line is the reference level for the lower
limit of retinal structure thickness at P31 (22SD from the mean at this age); photoreceptor structure above this lower limit indicates no difference
compared to the data of P31 (error bars, +2SD from mean).
CEP290 LCA Mouse
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PLOS ONE | www.plosone.org12 March 2014 | Volume 9 | Issue 3 | e92928