Identifying photoreceptors in blind eyes caused
by RPE65 mutations: Prerequisite for human
gene therapy success
Samuel G. Jacobson*†, Tomas S. Aleman*, Artur V. Cideciyan*, Alexander Sumaroka*, Sharon B. Schwartz*,
Elizabeth A. M. Windsor*, Elias I. Traboulsi‡, Elise Heon§, Steven J. Pittler¶, Ann H. Milam*, Albert M. Maguire*,
Krzysztof Palczewski?, Edwin M. Stone**, and Jean Bennett*
*Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104;‡Cole Eye Institute, Cleveland Clinic Foundation,
Cleveland, OH 44195;§Department of Ophthalmology and Vision Sciences, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8;
¶Department of Physiological Optics, University of Alabama at Birmingham, Birmingham, AL 35294;?Department of Ophthalmology,
University of Washington School of Medicine, Seattle, WA 98195; and **Howard Hughes Medical Institute and Department of
Ophthalmology, University of Iowa Carver College of Medicine, Iowa City, IA 52242
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 21, 2005 (received for review
January 25, 2005)
Mutations in RPE65, a gene essential to normal operation of the
visual (retinoid) cycle, cause the childhood blindness known as
Leber congenital amaurosis (LCA). Retinal gene therapy restores
vision to blind canine and murine models of LCA. Gene therapy in
blind humans with LCA from RPE65 mutations may also have
potential for success but only if the retinal photoreceptor layer is
intact, as in the early-disease stage-treated animals. Here, we use
high-resolution in vivo microscopy to quantify photoreceptor layer
thickness in the human disease to define the relationship of retinal
structure to vision and determine the potential for gene therapy
success. The normally cone photoreceptor-rich central retina and
rod-rich regions were studied. Despite severely reduced cone
vision, many RPE65-mutant retinas had near-normal central micro-
structure. Absent rod vision was associated with a detectable but
thinned photoreceptor layer. We asked whether abnormally
thinned RPE65-mutant retina with photoreceptor loss would re-
spond to treatment. Gene therapy in Rpe65?/?mice at advanced-
disease stages, a more faithful mimic of the humans we studied,
showed success but only in animals with better-preserved photo-
receptor structure. The results indicate that identifying and then
targeting retinal locations with retained photoreceptors will be a
prerequisite for successful gene therapy in humans with RPE65
mutations and in other retinal degenerative disorders now moving
from proof-of-concept studies toward clinical trials.
visual cycle ? Leber congenital amaurosis ? rod ? cone ? retinal imaging
mechanistic understanding and provided the hope of gene-based
therapies. One autosomal recessive human disease seems espe-
cially approachable with gene replacement therapy: the child-
hood-onset form of blindness known as Leber congenital am-
aurosis caused by mutations in the RPE65 gene (1). RPE65
encodes a 65-kDa protein located in the retinal pigment epithe-
lium (RPE) and essential for vertebrate vision. RPE65 mutations
prevent the normal cycling of retinoids, leading to photorecep-
tors without light-sensitive visual pigment and eyes with blind-
ness (1–8). Proof-of-concept studies using viral vector-mediated
RPE65 gene delivery to the eye have shown dramatic restoration
of vision in a naturally occurring canine model (9, 10) and a
murine knockout (11). Advance to human clinical trials would
seem to be the logical next step.
Translation from laboratory to clinic of gene therapy for
RPE65-associated Leber congenital amaurosis rests on the un-
proven assumption that the human disease shares a key feature
with the canine and murine models. The RPE65-mutant dog and
Rpe65?/?mouse show the unusual feature of dissociation of
iscovery of the molecular causes of blinding incurable
retinal degenerative diseases has improved diagnosis and
retinal structure and function. In most other retinal degenera-
tions, loss of photoreceptor structure is the underlying basis for
loss of photoreceptor function (12, 13). Both animal models of
RPE65 disease, however, retain photoreceptor structure despite
severe visual impairment, and the gene therapy successes have
occurred at disease stages when photoreceptor structure was still
How can it be determined whether humans with this genetic
blindness have photoreceptor structural integrity short of per-
forming retinal biopsy (14) or examining rare postmortem donor
tissue (15)? We used in vivo high-resolution microscopy and
correlative measures of vision (16–18) in patients with RPE65
mutations to determine the relationship of structure to function.
Photoreceptor layer thickness and vision were quantified in
regions normally rich in cones and rods. The finding of patches
of thinned photoreceptor layer in human RPE65 retinae led to
a study of gene therapy in late-stage Rpe65?/?mice with
photoreceptor loss. From these human and murine results
emerge guidelines for conducting a gene therapy trial in humans
with RPE65 mutations.
Materials and Methods
Human Subjects. Of the 59 participating patients, 11 had RPE65
mutations (Table 1). There were 48 patients (ages 9–68 years)
with negative RPE65 mutation screening (19) or X-linked or
dominant retinitis pigmentosa. Normal subjects (n ? 21, ages
procedures followed institutional guidelines and the Declaration
Optical Coherence Tomography (OCT). Cross-sectional retinal re-
flectivity profiles were obtained with OCT (Humphrey Instru-
ments, Dublin, CA) by using published techniques (16–18). In
eight RPE65 patients [all but patient 1 (P1), P2, and P11] and five
normals, six scan groups were used to cover a 18 ? 12-mm2
region of the retina centered on the fovea at high lateral
resolution. Each scan group covered a region of 6 ? 6 mm2of
retina by using 21 parallel horizontal (‘‘raster’’) scans of 6-mm
length, vertically separated by 0.3 mm (18). Postacquisition
processing of OCT data was performed with custom programs
(16–18). Spatial maps of retinal thickness were derived from the
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ERG, electroretinogram; OCT, optical coherence tomography; ONL, outer
nuclear layer; Pn, patient number; RPE, retinal pigment epithelium; Tx1, treatment group
1; Tx2, treatment group 2 .
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
April 26, 2005 ?
vol. 102 ?
no. 17 ?
groups of raster scans (18). Individual normal thickness maps
were aligned for fovea and optic nerve to determine local
statistics. Patient maps were aligned to normal maps and sub-
retinal regions of significant thinning. Cross sections of scans are
displayed after averaging two to five aligned scans and using a
2 ? 1 moving median filter to reduce speckle noise in lateral
dimension while keeping the original resolution in the longitu-
dinal direction. The location of the outer nuclear layer (ONL) in
cross-sectional scans was defined as the signal trough delimited
by the signal peaks corresponding to the outer plexiform layer
and outer limiting membrane. ONL thickness was defined in a
semiautomated fashion between the samples representing the
maximum slope on both sides of the signal trough (18).
Psychophysics. Dark-adapted thresholds were measured (at 2°
intervals, 650- and 500-nm stimuli, 1.7° diameter, 200-ms dura-
tion) in the same retinal regions as OCT scans. Visual function
techniques and analysis methods were as described (17, 18, 20).
Structure vs. Function. The relationship between photoreceptor
structure and colocalized visual function was defined in patients
by using ONL thickness and dark-adapted sensitivity. Patient
results were compared with an idealized model of the expected
relationship in pure photoreceptor degenerations. The model
assumes that photoreceptor function is proportional to the
product of the number of surviving photoreceptors and the
length of their outer segments; both of these parameters are
proportional to ONL thickness (13). Thus, to a first approxima-
tion, loss of light sensitivity (in linear units) would be expected
to be proportional to the square of ONL thinning.
Murine Studies: Animals and Experimental Procedures. Rpe65?/?
(n ? 82) and WT (n ? 12) mice of the same background (4) were
raised in 12-h on?12-h off cyclic dim (?3 lux) light. Treatment
unilateral subretinal injections of AAV2?1-CMV-hRPE65 (11).
Contralateral subretinal injections of buffered saline served as
controls. Treatment group 2 (Tx2; ages 17–26 mo, n ? 24) was
given 9-cis-retinal by oral gavage (5). Electroretinograms
(ERGs) were recorded ?2 mo after subretinal injections (n ?
25) and 48 h after oral cis-retinoid (n ? 21); untreated Rpe65?/?
mice (ages 3 mo, n ? 11; 16–24 mo, n ? 12) and WT mice (ages
3 mo, n ? 6; 15–25 mo, n ? 6) served as controls. Eyes were
enucleated for morphometry (Tx1: n ? 5; untreated Rpe65?/?:
n ? 7; WT: n ? 3) or retinoid analysis (Tx1: n ? 17; Tx2: n ?
22; untreated Rpe65?/?: n ? 10). Studies were in accordance
with the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Vision
Research and institutional review.
and 0.5% glutaraldehyde in 0.1 M PBS. Samples were washed in
Richardson’s mixture of methylene blue and azure II and photo-
graphed. Images were scaled by using a calibration tool photo-
graphed under the same (?40) magnification. ONL thickness
measurements were made at three locations separated 50 ?m from
each other and centered 1 mm from the optic nerve in superior
retina. An average of three measurements provided a single value
of ONL thickness for each retina.
Retinoid Analyses. All procedures related to extraction, derivati-
zation, and separation of retinoids from dissected mouse eyes
were as described (5, 22, 23). Each eye from treated animals was
analyzed individually. The following criteria were used to define
success in the unilateral subretinal AAV2?1-CMV-hRPE65-
treated animals (Tx1): the peak area of syn-11-cis-retinal oxime
was equal to or higher than 6 pmol, the peak elution for
syn-11-cis-retinal oxime was eluted ?0.3 min from the authentic
standard syn-11-cis-retinal oxime, and the untreated eyes did not
have the syn-11-cis-retinal peak. The following criteria were used
to define success in 9-cis-retinal-treated animals (Tx2): the
chromatographic separation of retinoid yielded absorbance at
325 nm for a peak corresponding to syn-9-cis-retinal oxime ?0.3
mAu, the results were comparable between two samples from
the same animal, both syn- and anti-9-cis-retinal oximes were
clearly separated, and the spectrum of syn-9-cis-retinal yielded a
low noise spectrum for the identification of the retinoid.
Retinal Thickness Topography in RPE65-Mutant Human Retinas. En
face viewing is the traditional method of assessing retinal ab-
normality in the clinic. The advent of high-resolution depth
(cross-sectional) imaging permits topographical maps of retinal
thickness to be constructed and disease effects to be analyzed by
this nontraditional metric (17, 18). As a step toward understand-
ing disease severity in human RPE65 mutations, we compared
the retinal thickness topography of the patients with that of
normal subjects (Fig. 1, en face views, Upper Insets). The retina
of a normal subject (age 22) has a central depression or foveal
pit, a surrounding ring of increased thickness with displaced
inner retinal layers from foveal formation, then, a decline in
shaped thickening extending into superior and inferior poles of
the optic nerve, attributable to the converging axons from
ganglion cells (Fig. 1A). The calculated lower limit of normal
thickness is also shown (Fig. 1A Lower Inset).
Retinal topography in a 20-year-old with RPE65 mutations, P5
(Fig. 1B), appears similar to normal. A retinal thickness difference
map (Fig. 1B Lower Inset) relates this RPE65-mutant retina to the
lower limit of normal and confirms that there are no areas of
reduced thickness. P4 (age 19) also has a normal retinal thickness
map (data not shown). A 21-year-old, P6 (Fig. 1C), retains topo-
graphical detail but the retina is thinned compared with normal.
The difference map highlights regions of significantly reduced
thickness; most prominent thinning is in a region ?1–3 mm from
the preserved central island. P7, a 27-year-old, shows retinal
thinning that is more widespread and extends into the temporal
retina (Fig. 1D). P9 and P10, ages 40 and 41, respectively, show
differences in retinal topography (Fig. 1 E and F). P9 has more
central retinal preservation of thickness than P10. The en face
fovea in P10 but not in P9. P8, at age 28, has similar topographical
and difference maps (data not shown) to those of P9. A range of
thickness topographies, suggesting a range of severities of retinal
Table 1. RPE65 mutations in Leber congenital amaurosis patients
PatientAge, y GenderMutationsSource*
IVS1 ? 5G?A, homoallelic
5, 17, 35, 48
*Previous report of genotype and?or phenotype.
www.pnas.org?cgi?doi?10.1073?pnas.0500646102Jacobson et al.
degeneration, thus exists among adults with RPE65 mutations and
there is no simple relationship of age and retinal thickness.
Photoreceptor Layer Is Definable in Adults with RPE65 Mutations.
Morphometry of the photoreceptors or ONL as thickness or cell
numbers has classically been used to assay retinal phenotype in
animal retinal degenerations (24, 25). Such histopathological data
have not been available for human retinopathies except from
postmortem donor tissue (12). Human ONL thickness has recently
become measurable in vivo by high-resolution optical cross-
sectional imaging (17, 18). We quantified ONL thickness along
horizontal and vertical meridia in normal and RPE65-mutant
retinas (Fig. 2). The normal human retina in cross section has
discernible laminae (Fig. 2A). At the cone-rich fovea, a reflectance
marks the vitreoretinal interface, a drop in reflectivity marks the
ONL, and deeper reflections correspond to photoreceptor inner
and outer segments and RPE. Temporal to the fovea in the
horizontal meridian (Fig. 2A Left) and also in the vertical meridian
(Fig. 2A Right), total retinal thickness increases and inner retinal
laminae are prominent. At further eccentricities, thickness gradu-
ally tapers. In the vertical meridian, in addition to neural and
synaptic laminae, ganglion cell axons traverse superficially to con-
retina, the ONL normally declines in thickness.
RPE65 mutant retinas also show an identifiable photoreceptor
lamina in cross section (Fig. 2 B–D). The foveas of P4 and P6
have normal ONL thickness, but P7 has abnormally reduced
thickness. Eccentric to the fovea, P4 and P6 show a more
pronounced decline in ONL thickness than normal. The ONL in
P4 is detectable and appears relatively constant in thickness
across horizontal and vertical meridia, but P6 shows an increase
in ONL thickness between ?3 and 6 mm in both temporal and
superior retinal sections. At greater eccentricities in the tempo-
ral retina, there is a decline in ONL thickness and it is not visible
at more superior retinal eccentricities. In P7, the ONL is not
evident beyond the very central retina.
Photoreceptor layer thickness for normal subjects and the 11
RPE65-mutant retinas was analyzed in horizontal (temporal) and
vertical (superior) meridia (Fig. 2E). At the fovea, there was
measurable ONL in all RPE65-mutant retinas and nearly half had
normal thickness. Immediately adjacent to the fovea, there was
abnormally reduced ONL and in many it was not detectable. An
increase in ONL thickness in the superior retina between ?3 and
6 mm from the fovea was evident in three retinas (P2, P4, and P6).
P6 also showed this increase in temporal retina (Fig. 2E).
RPE65-Mutant Retinas Have Greater Photoreceptor Layer Thickness
than Predicted for Amount of Visual Loss. The relationship between
photoreceptor nuclear layer structure and visual function was
examined in RPE65-mutant retinas at selected locations known to
have the highest densities of cones or rods in normal retinas (26).
Comparisons were made with normal subjects and other retinal
degenerations not caused by RPE65 mutations (Fig. 3). At the
fovea, average normal peak cone density is ?200,000 cells?mm?2
(26) and foveal ONL thickness in normal subjects averages 97 ?m
ONL thickness reduction was predictably related to central visual
function over a 3-log unit range from normal to severely abnormal
vision (Fig. 3A). All 11 patients with RPE65 mutations had abnor-
mally reduced foveal cone vision; 5 of 11 had no measurable
localized retinal thinning. Topographical maps from high-resolution depth
imaging of the central retina in a normal subject, age 22 (A), and five patients
(A Inset Lower Right) The lower limit (mean ? 2 SD) of normal. (B–F Insets
Lower Right) A difference map (subtracted from normal lower limit) showing
nerve. F, fovea. N, nasal, T, temporal, S, superior, I, inferior.
RPE65-mutant human retinas with normal thickness topography or
sectional retinal images along the horizontal (Left) and vertical (Right) me-
ridia through the fovea in a normal subject (A) and three patients with RPE65
mutations (B–D). ONL is indicated to the left of the images. (E) ONL thickness
across horizontal (Left) and vertical (Right) meridia in normal subjects and
SD (gray) are indicated.
Photoreceptor nuclear layer in RPE65-mutant retinas. (A–D) Cross-
Jacobson et al.PNAS ?
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expected for the level of dysfunction; in 5 individuals, ONL was
within normal limits. The relationship of function and structure in
three patients with RPE65 mutations was not distinguishable from
that of non-RPE65 patients.
Two rod-rich regions analyzed were at 3.6 mm eccentric to the
fovea. At 3.6 mm temporal to the fovea, rod photoreceptor
density normally peaks at ?140,000 cells?mm?2and the rod?
cone ratio is 20:1 (26). Normal ONL thickness at this location
averages 53 ?m (SD ? 6 ?m, n ? 17, age ? 19–56). The 3.6-mm
superior location is theoretically within the 3- to 5-mm eccen-
occur in normal human retina. Rod densities in this region
normally average 160,000 cells?mm?2and the rod?cone ratio is
?25:1 (26). Normal ONL thickness at this locus averages 56 ?m
(SD ? 6 ?m, n ? 14, age ? 19–56).
In non-RPE65 patients, ONL thickness reduction at the
rod-rich retinal loci was predictably related to dark-adapted
vision over a 5-log unit range from normal to severely abnormal
(Fig. 3 B and C). No vision was measurable in all 11 individuals
with RPE65 mutations at these loci. At the temporal location, 4
of 11, and at the superior location 5 of 11 RPE65-mutant retinas
showed a significantly greater amount of ONL thickness pres-
ervation for this severity of visual loss.
Rpe65?/?Mice at Late-Disease Stages Can Show Visual Restoration by
Gene Therapy. Given the evidence for abnormally reduced ONL
thickness (particularly outside the fovea) in many humans with
RPE65 mutations, we asked whether restoration of vision would
be possible at disease stages with significant photoreceptor
degeneration. To begin to answer this question, results of gene
therapy would need to be studied in an RPE65-mutant animal
with considerable photoreceptor loss. Natural history data in
Rpe65?/?mice indicate there is degeneration that progresses to
severe photoreceptor loss by 18–24 mo (27). We maintained
Rpe65?/?mice until older ages and treated them with subretinal
gene therapy; success rates for visual restoration at late- and
early-disease stages were then compared (11).
Retinal function in 3- to 4-mo-old Rpe65?/?mice is severely
units brighter stimuli to elicit responses than in WT mice (Fig.
4A). Gene therapy with AAV2?1-CMV-hRPE65 in Rpe65?/?
mice at ages 1–2.5 mo is ?80% successful by ERG assay 2 mo
later (11). A representative example of a therapeutic success
(treated at 2 mo, evaluated at 4 mo) shows dramatically im-
proved function with near-normal thresholds in the treated eye,
compared with the contralateral saline-injected control eye.
Gene therapy in Rpe65?/?mice at ages 17–24 mo (n ? 25) was
effective but in a far smaller percentage of eyes. Representative
ERGs from a 17-mo-old mouse with therapeutic success show
improved b-wave thresholds in the treated eye; the brightest
stimuli, however, did not elicit certain waveform features (e.g.,
a-wave) typically seen in WT (see age-related normal) or treated
younger Rpe65?/?mice (Fig. 4A).
layer than predicted from vision. (A) Foveal ONL thickness as a function of
dark-adapted cone-mediated sensitivity (650 nm). (B and C) ONL thickness as
a function of dark-adapted sensitivity (500 nm) at 3.6 mm in temporal (B) and
superior (C) retina. Rod, rod-mediated sensitivity; Cone, cone-mediated sen-
sitivity; Pts, patients without RPE65 mutations. Normal variability is described
by the ellipses encircling the 95% confidence interval of a bivariate Gaussian
distribution. Dotted lines define the idealized model of the relationship
region of uncertainty that results by translating the normal variability along
the idealized model. The region encompassing data with greater than ex-
pected ONL thickness is marked as treatment (Tx) potential. (Inset) Retinal
location (white arrow on fundus image) of colocalized measures of structure
and function. Overlaid onto the fundus image are cone density (a) and rod
density (b and c) along horizontal and vertical meridia (26).
RPE65-mutant human retinas can have more photoreceptor nuclear
limited visual restoration. (A) Comparison of ERGs in young (4 mo) and old
(?15 mo) WT (Left) and Rpe65?/?mice (Center and Right). (Center) Saline-
injected control eyes of Rpe65?/?mice illustrate the severe ERG abnormality.
(Right) The AAV2?1-CMV-hRPE65-treated eyes show visual restoration. Stim-
of treatment results. ERG thresholds in ?15-mo-old Rpe65?/?mice after
treatment with AAV2?1-CMV-hRPE65 (Tx1) or oral 9-cis retinal (Tx2) are
are results falling beyond the 99% confidence interval limit (upper boundary
of gray) determined from uninjected, age-matched, Rpe65?/?mice. Fre-
in young (empty bars) versus old (filled bars) Rpe65?/?mice is shown. (C) ONL
thickness (1 mm superior to optic nerve) in 3-mo-old vs. 24-mo-old Rpe65?/?
mice (F) is compared with age-matched WT (E) mice. ONL thickness data
(mean ? 2 SD) for young WT mice (25) is shown. (D) Retinal histological
sections (1 mm superior to optic nerve) from an old WT mouse (Left) are
compared with two old Rpe65?/?mice treated with gene therapy: one with
ERG success (Center) and one with failure (Right). The ONL and the inner
nuclear layer (INL) are indicated by brackets.
Gene therapy in Rpe65?/?mice at advanced disease stages leads to
www.pnas.org?cgi?doi?10.1073?pnas.0500646102Jacobson et al.
B-wave thresholds in control eyes were compared with their
contralateral-treated eyes (Fig. 4B, Ctrl vs. Tx1). Treatment
success was defined as results falling beyond the 99% confidence
interval limit (gray area) for b-wave threshold in uninjected,
age-matched Rpe65?/?mice. Success occurred in 4 of 25 animals
(16%). Thresholds in animals with a treatment effect were
significantly better than those from uninjected, age-matched,
Rpe65?/?(?1.65 ? 0.42 vs. ? 0.47 ? 1.45 log scot-cd?m?2?s??1)
mice (P ? 0.05) and fall within the level of improvement we
reported for young mice (11). The retinoid content was mea-
sured in treated eyes and saline-injected contralateral eyes from
a subset of the older animals. Five of 17 treated eyes (29%) had
clearly detectable 11-cis retinal; no measurable chromophore
was found in saline-injected control eyes (Fig. 4B).
Was the reduced success of treatment in older versus younger
mice (Fig. 4B Lower) possibly caused by subretinal surgical
intervention in these eyes with advanced retinal degeneration?
Oral 9-cis-retinal also successfully restores visual function in
?80% of treated young Rpe65?/?mice (5, 28) and does not
involve retinal surgery (Fig. 4B, Tx2). In a series of older mice
(n ? 21) of comparable ages to those used for gene therapy, oral
9-cis retinal was administered and ERGs were studied 48 h later.
These mice also had a lower success rate (24%) than younger
counterparts (Fig. 4B Lower); the success rates with oral inter-
vention versus gene therapy were not significantly different (P ?
0.05). Analysis of retinoid biochemistry in 9-cis-retinal-fed ani-
mals (n ? 22) indicated that treatment success rates were also
low. Only 6 of 22 (27%) mice had significant accumulation of
9-cis-retinal, whereas the rest of the treated mice had trace
amounts. Control (untreated) Rpe65?/?mice (n ? 10) did not
contain measurable 9-cis-retinal.
ONL thickness measurements in 3- and 17- to 24-mo-old
Rpe65?/?mice (Fig. 4C; retinal locus is 1 mm superior to optic
nerve) confirm previous data indicating that the ONL can be
age group, the ONL could have three to four photoreceptor
nuclei or be reduced to a single row, suggesting variability in the
amount of degeneration. We had the opportunity to inquire in
one of the gene therapy successes whether there was any
difference in ONL thickness compared with eyes that failed to
respond. Fig. 4D compares histological sections (at 1 mm
superior to the optic nerve) taken from an older WT mouse and
two treated Rpe65?/?mice. The eye with gene therapy treatment
success shows about four rows of nuclei, compared with the
treatment failure, which has only a single row of nuclei (similar
severity was found in three other treatment failures). This
with higher numbers of retained photoreceptor cells (and intact
RPE) are likely to have greater potential for treatment success.
Restoration of vision is the ultimate goal of human retinal degen-
eration research. In vivo gene transfer to photoreceptor and RPE
cells in animal models has led to dramatic results showing reversal
of defective gene function or prevention of apoptotic cell death (9,
29–32). This success has heightened expectation that the ultimate
goal is approachable in human eye disease. Specifically, for blind-
ness caused by RPE65 mutations, large and small animal models
been restored to blind animals that had mostly intact photorecep-
tors but lacked the 11-cis-retinal chromophore caused by an inter-
rupted retinoid cycle in the RPE.
Humans with RPE65 deficiency have been suspicious for
having complexity of disease mechanism: there is extremely
reduced photoreceptor function and visual loss from early life,
but there can be hallmarks of pigmentary degeneration and
atrophy of the retina (2, 3, 33–40). The reduced vision is
consistent with two possible disease mechanisms or a combina-
tion thereof: (i) an interrupted retinoid cycle that is potentially
treatable by RPE65 gene replacement, RPE cell replacement,
supplemental cis-retinoids, or any means to reestablish the
biochemical pathway (1), and (ii) loss of photoreceptors, which
would be more appropriate for therapeutic strategies such as
visual prosthetics (41). The relative contributions of pure dys-
function and cell death to the extreme visual loss in human
RPE65 deficiency are unknown. In a cohort of human retinal
degeneration patients without RPE65 mutations, we used colo-
calized in vivo measures of function (by visual thresholds) and
photoreceptor layer structure (by high-resolution cross-sectional
optical imaging) and established the relationship of visual loss to
cell loss. These data served as standards for comparison with
similar results from individuals with RPE65 mutations. Human
RPE65 disease did show a complex but interpretable structure–
function relationship that differs not only from retinal degen-
erations not caused by RPE65 mutation but also from the disease
stages in animal models when gene therapy was successful.
Our results for the cone-rich human fovea, a unique feature
of primates, are without comparable data from murine and
canine RPE65-mutant animals. Cone function, albeit severely
impaired, was detected centrally in nearly half of the subjects
with RPE65 mutations. Chromatic testing indicated that the
function was indeed cone-mediated. Disproportionate preser-
vation of foveal structure to a degree greater than expected for
the level of foveal cone function (Fig. 3A) suggests an effect of
RPE65 deficiency on human cones like that expected for rods.
The exact role of RPE65 in relation to cones, however, may be
more complex than that for rods (42, 43). The retinoid cycle of
cones and rods has differences (44). RPE65, originally identified
only in RPE, has also been found in cones of amphibians and in
mammalian retina, including cow, rabbit, and mouse (43). Fur-
ther complexity comes from a report of early cone degeneration
in Rpe65?/?mice (45). Our findings suggest that human foveal
cones may be more resistant to degeneration caused by RPE65
deficiency and thus most amenable to gene replacement therapy.
Rod photoreceptor topography in humans is not uniform, and
there is a ring of highest rod density at ?3–5 mm from the fovea
(26). The rod?cone ratio in this region of human retina approxi-
mates that in murine and canine retina. Structure and function
analyses in RPE65-deficient humans showed examples with no
difference in function–structure pattern to other retinal degener-
ations, but there were also dramatic examples of greater ONL
thickness than predicted from the level of impaired visual function.
suggest greatest preservation of the photoreceptor lamina at the
region of highest rod density, i.e., 3- to 5-mm eccentricity (26). The
region of reduced ONL thickness at lesser eccentricities resides on
shape of the ONL plots in RPE65-mutant retinas is that they
represent cone and rod spatial densities. Peak ONL at the fovea
reflects the peak spatial density of cones, which are retained in
many RPE65-deficient retinas. The reduced ONL in extrafoveal
retina may represent cone nuclei and?or a severely reduced spatial
density of rods. The increased ONL thickness at 3- to 5-mm
eccentricity, the site of the rod hot spot (26), suggests the presence
of residual rod nuclei.
disease sufficiently resembles the animal models to warrant gene
therapy in humans, the answer is in the affirmative but with
caveats. In the adult RPE65-mutant retinas we studied, extrafo-
veal retinal regions with anatomical preservation would seem
justifiable to treat. The implicit assumption is that measurable
ONL has adjacent functional RPE (not measurable exclusive of
other substructures by using OCT). The results of treatment of
the hypothesis that degenerate retina would be treatable given
enough retinal and RPE integrity. Our finding of variable
Jacobson et al. PNAS ?
April 26, 2005 ?
vol. 102 ?
no. 17 ?
degrees of surviving photoreceptors at late disease stages in Download full-text
inherited retinal degeneration is not novel; other late-stage
inherited retinal degenerations show similar variability (46). Far
less success of gene therapy at later disease stages points to the
intuitive notion that a thicker ONL would have more potential
for functional recovery. A key concept from thickness topogra-
phy and ONL measurements in these adult RPE65-deficient
humans is that there was no straightforward relationship of age
and amount of retinal degeneration.
Gene therapy targeted at the pericentral retina to restore rod
(and cone) function and?or at the fovea to restore cone function
thus seem worthy strategies, if pretreatment imaging studies indi-
cate potential value of therapy. Focal intervention at exact retinal
locations identified by pretreatment studies has been routine for
many other retinopathies. For example, pretreatment angiography
guides laser application to destroy abnormal subretinal angiogen-
esis and surgical removal of such vessels (47). Whether infants and
children with RPE65 mutations have less abnormal photoreceptor
structure and require less pretreatment planning needs to be
determined as human ocular gene therapy to restore vision
progresses from a hopeful goal to an achievement.
We thank T. Redmond, E. Smilko, S. Hagstrom, M. Batten, A. Roman,
Grants EY009339, EY013385, EY013729, and EY013203; the Founda-
tion Fighting Blindness; the Macula Vision Research Foundation; the
F. M. Kirby Foundation; the E. K. Bishop Foundation; the Macular
Disease Foundation; Research to Prevent Blindness; and the Mackall
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