Gene therapy for retinitis pigmentosa caused by MFRP mutations: human phenotype and preliminary proof of concept.
ABSTRACT Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.
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ABSTRACT: Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice-an established preclinical model of RP-and long-term improvement in visual function was observed in AAV-Mfrp treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.Molecular Therapy (2014); doi:10.1038/mt.2014.100.Molecular therapy : the journal of the American Society of Gene Therapy. 06/2014;
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ABSTRACT: Purpose: We present a genetic and clinical analysis of two sisters, 3 and 4 years of age, with nanophthalmos and macular folds. Methods: Ophthalmological examination, general paediatric examination and molecular genetic analysis of the MFRP gene were performed in both affected siblings. Results: Clinical analysis showed high hyperopia (+11 D and +12 D), short axial lengths (15 mm) and the presence of macular folds and optic nerve head drusen. Autofluorescence of the retina was generally normal with subtle macular abnormalities. Sequence analysis showed compound heterozygosity for severe MFRP mutations in both sisters: a previously reported p.Asn167fs (c.498dupC) and a novel stop codon mutation p.Gln91X (c.271C>T). Conclusion: These are the youngest nanophthalmos patients in the literature identified with severe loss of MFRP function, showing already the known structural abnormalities for this disease. Adult patients affected by homozygous or compound heterozygous MFRP mutations generally show signs of retinal dystrophy, with ERG disturbances and RPE abnormalities on autofluorescence imaging. ERG examination could not be performed in these children, but extensive RPE abnormalities were not seen at this young age.Acta ophthalmologica 06/2013; · 2.44 Impact Factor
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ABSTRACT: Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects, increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they all frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller cells and microglia, because their response to injury is decisive for maintaining the health of the retina or its degeneration. Several therapeutic approaches have been developed to preserve retinal function or restore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy, cell transplantation or artificial devices should be applied at the appropriate stage of retinal degeneration to obtain successful results. This review provides an overview of the common and distinctive features of retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused by the cellular response to damage, in order to establish appropriate treatments for these pathologies.Progress in Retinal and Eye Research 01/2014; · 9.44 Impact Factor
Gene Therapy for Retinitis Pigmentosa Caused
by MFRP Mutations: Human Phenotype
and Preliminary Proof of Concept
Astra Dinculescu,1Jackie Estreicher,2Juan C. Zenteno,3Tomas S. Aleman,2Sharon B. Schwartz,2
Wei Chieh Huang,2Alejandro J. Roman,2Alexander Sumaroka,2Qiuhong Li,1Wen-Tao Deng,1
Seok-Hong Min,1Vince A. Chiodo,1Andy Neeley,1Xuan Liu,1Xinhua Shu,4
Margarita Matias-Florentino,3Beatriz Buentello-Volante,3Sanford L. Boye,1
Artur V. Cideciyan,2William W. Hauswirth,1and Samuel G. Jacobson2
Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due
to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mu-
tations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal
and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate
for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-
acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken b-actin promoter driving the
wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal de-
generation. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal
histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression
predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli.
The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6
mouse suggest that this form of RP is a potential target for gene-based therapy.
tion, and survival of photoreceptor cells (Sparrow et al., 2010).
On its apical side, the RPE extends numerous microvilli
around the light-sensitive photoreceptor outer segments and
into the interphotoreceptor matrix. Microvilli considerably
increase the surface area of the RPE apical membrane, and
establish a critical interface for many RPE functions including
phagocytosis of shed outer segments, visual chromophore
transport and regeneration, production of trophic and anti-
angiogenic factors, directional transport of oxygen and nu-
trients from the choroid to sustain the high metabolic rate of
photoreceptors, and removal of water and aqueous waste
he retinal pigment epithelium (RPE) plays a critical
role in vision, maintaining the structural integrity, func-
products from the subretinal space (Strauss, 2005; Bonilha
et al., 2006). Mutations in genes expressed in RPE cells can
2010). Some of these include RPE65 and LRAT (Leber con-
genital amaurosis, LCA), MERTK (early-onset retinitis pig-
mentosa, RP), BEST1 (Best disease), TIMP3 (Sorsby fundus
dystrophy), EFEMP1 (malattia leventinese), RDH5 (fundus
albipunctatus), and RLBP1 (retinitis punctata albescens).
A relative newcomer to this group is autosomal reces-
sively inherited RP caused by mutations in the human MFRP
(membrane-type frizzled related protein) gene, located on
chromosome 11q23 (Ayala-Ramirez et al., 2006; Crespı ´ et al.,
2008; Zenteno et al., 2009; Mukhopadhyay et al., 2010). The
MFRP gene encodes a type II transmembrane protein of 584
amino acid residues, which consists of an N-terminal
1Department of Ophthalmology, University of Florida, Gainesville, FL 32610.
2Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
3Biochemistry Department, Faculty of Medicine, National Autonomous University of Mexico (UNAM); and Department of Genetics-
Research Unit, Institute of Ophthalmology Conde de Valenciana, Mexico City, CP 06800, Mexico.
4Department of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, Scotland.
HUMAN GENE THERAPY 23:367–376 (April 2012)
ª Mary Ann Liebert, Inc.
cytoplasmic region, a transmembrane domain, and an ex-
tracellular region with a C-terminal cysteine-rich domain
similar to that observed in Wnt-binding frizzled proteins
(Katoh, 2001; Kameya et al., 2002). Mfrp is expressed as one
element of a dicistronic transcript (Kameya et al., 2002;
Hayward et al., 2003; Mandal et al., 2006), which also encodes
the complement C1q tumor necrosis factor-related protein-5
(C1QTNF5/CTRP5). CTRP5 also has a human disease asso-
ciation; specifically, a Ser163Arg mutation causes an auto-
somal dominant late-onset retina-wide degeneration, which
can feature neovascular macular degeneration (Milam et al.,
2000; Jacobson et al., 2001; Hayward et al., 2003). The func-
tional relationship between the two proteins remains under
investigation (Fogerty and Besharse, 2011).
With clinical trials of gene therapy ongoing for another
autosomal recessive RPE disease leading to retinal degener-
ation, RPE65-LCA (reviewed in Cideciyan, 2010), we in-
quired whether MFRP-RP was a potential candidate for this
form of treatment. A patient with MFRP-RP was examined in
detail by noninvasive studies and the results were compared
with those of patients with other molecularly proven MFRP-
RP in the literature. Like RPE65-LCA, there is an animal
model of the MFRP-RP disease for proof-of-concept studies.
The rd6 mouse has a naturally occurring autosomal recessive
retinal degeneration associated with a 4-bp deletion in a
splice donor site in the Mfrp gene (Hawes et al., 2000;
Kameya et al., 2002). This results in the skipping of exon 4
and deletion of 58 amino acids from the Mfrp protein
(Kameya et al., 2002). The present study uses subretinal
delivery of a self-complementary tyrosine-capsid mutant
adeno-associated virus serotype 8 (AAV8) (Y733F) vector
carrying the mouse Mfrp gene to determine whether photo-
receptor degeneration can be prevented in rd6 mice, thereby
paving the way for further proof-of-concept, dosing, and
safety studies in gene therapy en route to a clinical trial in
patients with MFRP-RP.
Materials and Methods
A patient with RP underwent a complete eye examination
and detailed studies of retinal phenotype. Genomic DNA
from peripheral blood lymphocytes was obtained from the
patient and family members and molecular genetic analyses
led to the identification of MFRP mutations (Ayala-Ramirez
et al., 2006; Crespı ´ et al., 2008; Zenteno et al., 2009). Patients
with retinal degenerations, including other forms of RP
(n=6; age, 7–78 years) and choroideremia (n=3; age, 13, 34,
and 38 years), as well as normal subjects (n=34; age, 5–58),
were included for comparison of phenotype. Informed con-
sent was obtained for all subjects; procedures were in ac-
cordance with the Declaration of Helsinki and were
approved by the institutional review board.
Human phenotype studies
Full-field electroretinograms (ERGs) were performed with
bipolar Burian-Allen contact lens electrodes and a standard
protocol using an Espion system (Diagnosys, Lowell, MA)
with methodology previously described (Jacobson et al.,
1989; Aleman et al., 2002). Kinetic visual fields and dark- and
light-adapted chromatic static threshold perimetry (200-msec
duration, 650- and 500-nm stimuli, dark-adapted, and
600nm, light-adapted; 1.7? diameter target) were performed
with a modified HFA-750i analyzer (Zeiss-Humphrey, Du-
blin, CA). Our methods for data collection and analyses have
been published (Jacobson et al., 1986, 2010; Roman et al.,
2005). Reflectance imaging and reduced-illuminance auto-
fluorescence imaging (RAFI) were performed with near-
infrared (NIR) and short-wavelength (SW) lights with a
confocal scanning laser ophthalmoscope (SLO) (HRA2; Hei-
delberg Engineering, Heidelberg, Germany) as described
(Cideciyan et al., 2007, 2011; Jacobson et al., 2011). Retinal
cross-sectional imaging used a spectral-domain optical co-
herence tomography (SD-OCT) unit (RTVue-100; Optovue,
Fremont, CA) with published recording and analysis tech-
niques (Cideciyan et al., 2011; Jacobson et al., 2011). The
three-dimensional SD-OCT raster scans were performed for
topographic analysis. Postacquisition processing of OCT
data was performed with custom programs (MATLAB 6.5;
MathWorks, Natick, MA). Further methodological details are
provided in the online supplement (supplementary data are
available online at www.liebertonline.com/hum).
rd6 mice (originally provided by B. Chang, Jackson La-
boratory, Bar Harbor, ME) were bred and maintained in the
University of Florida Health Science Center Animal Care
Services Facility (Gainesville, FL) under 12-hr-on/12-hr-off
cyclic lighting. Wild-type C57BL/6 mice served as controls
and were from the University of Florida or University of
Pennsylvania (Philadelphia, PA) Animal Care Services Fa-
cility. All experiments were approved by University of
Florida and University of Pennsylvania Institutional Animal
Care and Use Committees and conducted in accordance with
the Association for Research in Vision and Ophthalmology
(ARVO) Statement for the Use of Animals in Ophthalmic and
Recombinant AAV preparation and subretinal
delivery in mice
A self-complementary AAV8 vector containing a Y733F
point mutation in a highly conserved surface-exposed capsid
tyrosine residue was used for packaging the wild-type mu-
rine Mfrp cDNA under the control of the ubiquitous, con-
stitutive smCBA (small chicken b-actin) promoter. AAV8
(Y733F) vector was produced by the two-plasmid co-
transfection method in HEK 293 cells and purified according
to previously reported methods (Petrs-Silva et al., 2009). Viral
titer was determined by real-time PCR. Subretinal injections
were performed on postnatal day 14 (P14) under anesthesia
as described (Pang et al., 2008). In brief, the nasal cornea was
penetrated with a 30.5-gauge disposable needle; and a 33-
gauge unbeveled, blunt-tip needle on a Hamilton syringe
was introduced into the subretinal space. Each eye received
1ll of vector at a titer of 1·1012genome copies/ml, leaving
the left eye as an untreated control. Preliminary experiments
were also performed with buffer-injected rd6 eyes as possible
controls, and it was observed that there was rapid degen-
eration in these eyes, likely secondary to poor reattachment
after retinal detachment. To avoid bias that would lead to an
apparent treatment effect due to surgery-related damage to
control rd6 eyes, we chose to use an untreated control eye for
368DINCULESCU ET AL.
comparison with treatment. Eleven rd6 mice had subretinal
injections of vector in the right eye. At 6 weeks postinjection,
mice with no signs of injection-related trauma to the anterior
segment were included in further studies.
Electroretinography in mice
Full-field ERGs were elicited in 2-month-old (P14-treated)
rd6 mice (n=5) and wild-type C57BL/6 controls (n=20;
mean age, 3.4 months; age range, 1 to 10 months), using
methods previously reported (Roman et al., 2007; Pang et al.,
2008; Caruso et al., 2010). A series of stimuli were gener-
ated with a UTAS SunBurst system (LKC Technologies,
Gaithersburg, MD) or a custom-built ganzfeld stimulator
with a computer-based system (EPIC-XL; LKC Technolo-
gies). Mice were dark-adapted (>12hr) and anesthetized
by injection of a mixture of ketamine-HCl (72mg/kg) and
xylazine (5mg/kg). Pupils were dilated with topical agents
(phenylephrine hydrochloride, 2.5%; tropicamide, 1%) under
dim red light. Dark-adapted ERGs were elicited with 0.02
and 2 scot-cd$sec$m–2stimuli. In wild-type mice, the dimmer
flash produces a b-wave driven by rod postreceptoral ac-
tivity whereas the brighter flash produces an a-wave domi-
nated by rod photoreceptor activity and a b-wave that is driven
by both rod and cone postreceptoral neurons (Weymouth
and Vingrys, 2008). In addition, cone-isolated ERGs were
elicited with 25 phot-cd$sec$m–2stimuli on a rod-suppressing
background light after a preadaptation period. In wild-type
mice, this flash produces a b-wave driven by cone post-
receptoral neurons (Weymouth and Vingrys, 2008). Efficacy
of the uniocular treatment in rd6 eyes was determined by
evaluating interocular differences (IODs). IODs for all ERG
parameters were expressed as the amplitude difference be-
tween the two eyes divided by the mean value. t tests were
used to determine the statistical significance of differences
between IODs of rd6 mice and wild-type animals.
Retinal histology and immunostaining
For morphological analysis, treated rd6 mice (n=4) had
both eyes fixed in 10% formalin solution, processed for
paraffin embedding, sectioned at a thickness of 4lm, and
stained with hematoxylin and eosin. The sections were vi-
sualized by light microscopy. Comparisons were made with
adult C57BL/6 mice (n=4). The outer nuclear layer (ONL)
thickness was measured at three locations from within the
mid-superior retina of wild-type, treated, and untreated rd6
eyes (four animals per group). The differences between the
ONL thickness of AAV-treated and the uninjected contra-
lateral left eyes were analyzed by Student t test for paired
samples. A difference was considered significant at p<0.05.
For immunostaining, deparaffinized tissue sections were
dewaxed in xylene and rehydrated in a graded series of
ethanol, and then incubated with 0.5% Triton X-100 for
15min, followed by blocking with a solution of 2% albumin
for 30min. The sections were incubated with mouse MFRP
affinity-purified polyclonal antibody (AF3445, R&D Systems,
Minneapolis, MN) or anti-ezrin (Sigma-Aldrich, St. Louis,
MO). Secondary antibodies were Alexa-594 or Alexa-488
fluorophore (Molecular Probes/Invitrogen, Eugene, OR) di-
luted 1:500 in 1· phosphate-buffered saline (PBS). All sec-
tions were examined by fluorescence microscopy, using a
Leica TCS SP2 laser scanning confocal microscope (Leica,
Heidelberg, Germany). Albino control mouse retinas were
used as immunostaining controls to prevent the melanin in
the RPE from interfering with the signal.
Western blot analysis
AAV8 (Y733F)-smCBA-MFRP-treated and untreated rd6
eyes were carefully dissected, and the MFRP protein was
detected by Western blotting. The eyecups were homoge-
nized by sonication in 1· PBS containing complete protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).
After centrifugation, each pellet was resuspended in a buffer
containing 50mM Tris-HCl (pH 7.4), 1% Triton X-100, 2%
sodium dodecyl sulfate (SDS), and 10% glycerol, and used
for Western blot analysis. Aliquot extracts containing equal
amounts of protein were subjected to SDS–polyacrylamide
gel electrophoresis (PAGE), using a 4–12% gradient gel, and
transferred to polyvinylidene difluoride (PVDF) membranes
(Millipore, Bedford, MA). After incubation for 1hr in
Odyssey blocking buffer (Li-Cor, Lincoln, NE), the mem-
branes were probed either with a primary antibody against
MFRP (mouse MFRP AF3445; R&D Systems) or an antibody
against a-tubulin (rabbit polyclonal ab4074; Abcam, Cam-
bridge, MA) as an internal control. The signals were detected
with an infrared IRDye 800 dye-conjugated secondary anti-
body (Rockland Immunochemicals, Gilbertsville, PA). Vi-
sualization of specific bands was performed with the
Odyssey infrared fluorescence imaging system (Odyssey;
MFRP-RP human phenotype
A 19-year-old female patient had spectacle correction in
childhood, There were some complaints of night and pe-
ripheral vision disturbances as well as difficulty with read-
ing; these visual symptoms were noted mainly from the
second decade of life. Ancestry was German/English/
French with no known parental consanguinity; parents and
siblings had no visual complaints. Visual acuity was 20/30
with a refractive error of +11.00 sphere in each eye. Corneal
curvatures, ultrasound A-scan, and anterior chamber depth
were 52.00D sphericalequivalent
43.95–1.470) (AlMahmoud et al., 2011), 16.4mm (normal,
23.67–0.9) (Oliveira et al., 2007), and 2.2mm (normal,
2.9–0.3) (Fontana and Brubaker, 1980), respectively. Corneal
diameters and intraocular pressures were normal. Near-
infrared (NIR) reflectance images of the fundus showed
irregular margins of the optic nerve head, and peripheral reti-
nal regions with chorioretinal atrophy and bone spicule-like
pigment (Fig. 1A). Autofluorescence imaging with SW and
NIR excitation were consistent with a *20? diameter central
region of retained RPE with normal or nearly normal sig-
nals originating from lipofuscin and melanin fluorophores.
Surrounding the central region was an annulus of hyper-
autofluorescence apparent in both SW- and NIR-RAFI.
This annulus could originate from unmasking of existing
fluorophores, accumulation of additional fluorophores, or
chemical changes affecting their fluorescence efficiency.
Surrounding the hyperautofluorescent ring was an interme-
diate level of autofluorescence like that previously described
in other forms of RP (Cideciyan et al., 2011). Optic disc
OCULAR GENE THERAPY FOR MFRP-RP 369
imaging of the patient (top). Arrows point to bone spicule-like pigment. Melanin abnormalities are visible on reduced-
illuminance autofluorescence imaging (RAFI) with NIR light (bottom left) and lipofuscin abnormalities are demonstrated on
RAFI with short-wavelength (SW) light (bottom right). Insets: Representative normal images for each modality. Images are
individually contrast-stretched for visibility of features. (B) Electroretinography. Standard full-field elecroretinograms (ERGs)
from a normal subject and a patient with MFRP-RP. Rod b-waves were reduced to 4% of mean normal; mixed ERGs had
reduced a-waves (11% of normal) and b-waves (7% of normal); and cone ERGs (1 and 30Hz) were reduced to 35% and 10% of
normal, respectively. Stimulus onset was at the start of traces. Calibrations are shown to the right and below the waveforms.
(C) Psychophysics. Dark- and light-adapted static threshold perimetry results are displayed as grayscale maps of rod and
cone sensitivity loss. The physiological blind spot is shown as a black square at 12? in the temporal field. N, T, I, and S, nasal,
temporal, inferior, and superior visual field, respectively. Kinetic perimetry results (inset, top right) illustrate some visual field
constriction (nasally) for the larger target (V-4e) with 70% of normal extent, and a central island with 10% normal extent,
using the small target (I-4e). (D and E) Thickness topography of total retina, inner retina, and outer nuclear layer mapped in
the central retina for normal subjects (D, n=6; age, 21–41 years) and the patient with MFRP-RP (E). Insets in the lower right-
hand corner of patient data indicate whether the thickness measurements are within normal limits (white), abnormally thin
(blue, less than 2 SD), or abnormally thickened (pink, greater than 2 SD). (F–H) Retinal laminar architecture. Cross-sectional
optical coherence tomography (OCT) images along the horizontal meridian through the fovea in a normal subject (F) are
compared with those of a patient with MFRP-RP (G). Examples of OCT cross-sections in other retinopathies with abnormal
foveal shapes are shown along with measurements of the hyperreflectivity at the vitreoretinal interface in normal subjects;
patients with RP with cystoid macular edema (CME) and choroideremia (CHM); and the patient with MFRP-RP (H). Error
bars, 1 SD. MFRP-RP, MFRP (membrane-type frizzled-related protein) gene mutation-associated retinitis pigmentosa.
Human MFRP-RP phenotype. (A) En face imaging. Digitally stitched wide-field, near-infrared reflectance (NIR-REF)
drusen were detectable as small hyperautofluorescent dots
prompted questions concerning whether the patient had
MFRP-RP (Ayala-Ramirez et al., 2006; Crespı ´ et al., 2008;
Zenteno et al., 2009; Mukhopadhyay et al., 2010). Partial
nucleotide sequencing of MFRP exon 5 from the proband
showed a heterozygous 1-bp deletion (Supplementary Fig.
S1A; c.498delC, arrow) (supplementary data are available
online at www.liebertonline.com/hum), which predicts a
prematurely truncated protein (p.Asn167ThrfsX25). The
normal exon 5 sequence is shown (Supplementary Fig. S1B).
In the second allele a novel heterozygous G-to-T mutation
(Supplementary Fig. S1C, arrow) at the conserved 5¢ donor
splice site was demonstrated at exon/intron 9. The normal
exon/intron 9 sequence is also illustrated (Supplementary
Fig. S1D). Parental DNA analysis showed that the father
carried one allele with the one base deletion and the mother
carried one allele with the splice site mutation.
Full-field ERGs were abnormal (Fig. 1B). Rod b-waves
were barely detectable (11lV; normal, 299–52lV); a mixed
cone–rod ERG had reduced a- and b-wave amplitudes (a-
wave, 33lV [normal, 297–65lV]; b-wave, 35lV [normal,
497–111lV]); and cone ERGs were reduced in amplitude
(for single flash, 61lV [normal, 173–32lV]; and for flicker,
17lV [normal, 172–35lV]). A rod sensitivity loss map by
psychophysics showed no detectable rod function in the far
peripheral field, but there was some retained rod sensitivity
(between *1.5 and 2.5 log10units reduced) in a wide region
of the central field (Fig. 1C, left). Cone sensitivity loss was
most evident in the nasal field, but there was detectable cone
function elsewhere in the field. At fixation, cone sensitivity
was within normal limits but was reduced by 0.5–1.5 log10
units with increasing eccentricity into the temporal periph-
eral field (Fig. 1C, right). A kinetic visual field showed slight
generalized constriction (more evident in the nasal field) to
the V-4e target (70% of normal extent; 90% is 2 SD less than
the normal mean) (Jacobson et al., 1989); the I-4e target was
detected only centrally (10% of normal extent; 90% is 2 SD
less than the normal mean) (Fig. 1C, inset).
Topographical maps using OCT of retinal thickness and
maps of inner retina and photoreceptor outer nuclear layer
(ONL) are shown for normal subjects compared with the
patient with MFRP-RP (Fig. 1D and E). Whereas retinal
thickness was within normal limits across most of the retina
except in the very central macula, the inner retina was sig-
nificantly thicker than normal across the retinal area studied.
The ONL, on the other hand, was thicker than normal at the
fovea, within normal limits in the parafoveal region, but
thinner than normal across the remainder of the retina
sampled (Fig. 1D and E, insets, are comparison maps to
normal limits). Cross-sectional images revealed abnormal
retinal laminar architecture in the patient with MFRP-RP. A
normal fovea has a structural pit due to the concentric
displacement of an inner nuclear layer (INL) and limited
hyperreflective tissue vitreal to the cone outer nuclear layer
(Fig. 1F). In contrast, the MFRP-RP fovea was devoid of a
normal central depression and had substantial inner retinal
lamination with a discernable INL, microcystic changes an-
terior to the ONL, and considerable tissue vitreal to the INL
(Fig. 1G). The deep hyporeflective layer had no cystic
structures and measured 182.8lm (normal foveal ONL in the
central 0.6mm, 90.1–10.5lm; n=16; age, 11–28 years) (Ja-
cobson et al., 2007).
We then asked whether the inner and outer segment (IS/
OS) layer thickness was also different from normal in the
patient with MFRP-RP and found the thickness across the
central 4mm to be within normal limits (data not shown).
This would suggest that this hyperthick layer may be com-
plicated by Muller cell swelling, such as we suggested may
be the cause of a similar-appearing effect in patients with
choroideremia (CHM) (Jacobson et al., 2006). Thickness at the
fovea with concomitant loss of the foveal pit at certain stages
of CHM could reach 150–190lm, similar to that seen in this
patient with MFRP-RP. A more common cause of thickened
central structure in RP is cystoid macular edema (CME), but
no cysts were visible within the ONL layer scans of the pa-
tient with MFRP-RP. The thick hyperreflective inner retinal
layer across the foveal region in this patient measured
160lm, which is in dramatic contrast to the relatively thin
foveal hyperreflectivity at the vitreoretinal interface in nor-
mal subjects (9.8–1.5lm; n=7; age, 9–45 years). Patients
with CHM with hyperthick foveal hyporeflectivity and no
cystic changes (n=3; age, 13, 34, and 38 years) and patients
with RP with CME (n=6; age, 7–78 years) showed thicker
than normal hyperreflectivity at the vitreoretinal interface,
but the values were small (average, RP with CME, 34.8lm;
CHM, 30.7lm) by comparison with that in the patient with
MFRP-RP (Fig. 1H).
Retinal function and morphology in AAV
vector-treated rd6 mice
The natural history of retinal function in the rd6 mouse has
been reported to show a decrease in ERG amplitude detect-
able by P25 followed by a progressive decline (Hawes et al.,
2000; Won et al., 2008). Dark-adapted ERGs, dominated by
rod function, show greater reduction than light-adapted
ERGs, mediated by cone function, at early disease stages
(Won et al., 2008). Representative ERG waveforms driven by
the activity of rods, both rods and cones (mixed rod–cone),
and cones are shown for a wild-type mouse and for the
untreated and treated eyes of a 2-month-old rd6 mouse (Fig.
2A). At the ages studied, for rod b-wave amplitudes, the
mean of untreated rd6 eyes was 58% of mean wild-type,
whereas, for cone-isolated b-waves, the mean of untreated
rd6 eyes was 89% of mean wild-type. Despite the tendency
for lower values compared with wild-type, all rd6 eyes were
within normal limits for these parameters. For mixed rod–
cone ERGs, the majority (60%) of the untreated rd6 eyes had
abnormally reduced amplitudes with mean values being
reduced to 45 and 56% of mean wild-type for a- and b-
ERGs performed 6 weeks after the uniocular subretinal
injection of 1ll (109total vector genomes) of AAV8 (Y733F)
vector carrying wild-type mouse Mfrp cDNA showed there
were higher amplitudes in treated versus untreated rd6 eyes.
ERGs for all treated rd6 eyes were within normal limits. For
the representative rd6 mouse (Fig. 2A), the treated eye had a
rod-isolated b-wave amplitude of 462lV as compared with
225lV in the untreated eye (interocular difference, 69%).
Across all rd6 animals, treated eyes had larger amplitudes
(range, 316–462lV) compared with untreated eyes (160–
238lV) with interocular differences (treated minus untreated)
OCULAR GENE THERAPY FOR MFRP-RP 371
(WT) mouse eye compared with those from the two eyes of a 2-month-old rd6 mouse that was treated uniocularly with
vector–gene 6 weeks previously. Rod-isolated responses were elicited under dark-adapted conditions with dim (0.02 scot-
cd$s$m–2) flashes; mixed rod- and cone-driven responses were elicited with brighter (2 scot-cd$s$m–2) flashes. Cone-isolated
responses were evoked with 25 phot-cd$s$m–2stimuli on a rod-desensitizing background. (B) Interocular difference of four
ERG parameters plotted for individual rd6 animals (red circles) and wild-type mice (black squares). *p<0.01 for t tests
comparing the means of two groups of animals. L-R, left-right; T-U, treated-untreated. (C) Light microscopy of a 2-month-
old wild-type retina (left) and untreated (middle) and treated rd6 retinas (right). Note the shorter outer segments (OS), reduced
outer nuclear layer (ONL) thickness, and the presence of phagocytic-like cells (arrows) in the untreated rd6 eye. RPE, retinal
pigment epithelium; IS, inner segments; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 20lm.
Functional and structural consequences of gene therapy in rd6 mice. (A) Representative ERG traces from a wild-type
MFRP expression in wild-type albino control retinas (scale bar, 20lm). Note the increased distance between the outer nuclear
layer (ONL) and retinal pigment epithelium (RPE) [compared with (B) and (C)] caused by artifactual postmortem retinal
detachment. Inset: Image of the entire retina at lower magnification (scale bar, 0.5mm). (B) Untreated rd6 retina showing no
detectable MFRP expression. (C) Vector-treated rd6 retina section showing robust MFRP immunostaining predominantly in
the RPE apical membrane. Inset: Full retinal section at low magnification, showing the widespread area of transduction (scale
bar, 0.5mm). DAPI (blue) staining of nuclei. (D and E) Higher magnification of the RPE layer from (A) and (C), respectively,
depicting strong MFRP expression in the apical RPE membrane and its microvilli (arrowhead) (scale bars, 10lm). (F) Western
blot analysis of whole eyecup protein extracts from a 2-month-old untreated (U) rd6 eye and the partner vector-treated (T)
eye. Note the presence of an immunoreactive MFRP band in the treated eye only.
Immunolocalization of MFRP and Western blot analysis in rd6 mice with and without gene therapy. (A) Detection of
averaging 54% (range, 34–69%) as compared with 3% (range,
-21 to +31%) in wild-type mice (Fig. 2B). With the mixed
rod–cone ERGs, the treated rd6 eyes had larger amplitudes (a-
wave range, 132–265lV; b-wave range, 326–604lV) as com-
pared with untreated eyes (a-wave range, 47–147lV; b-wave
range, 153–369lV) with interocular differences averaging 83%
(range, 35–99%) for a-waves and 57% (range, 40–72%) for
b-waves. Wild-type mice for the mixed rod–cone ERGs had a
mean interocular difference near zero (range, -24 to +32%)
(Fig. 2B). For the cone-isolated ERG, the treated rd6 eyes also
had larger b-wave amplitudes (range, 129–175lV) as com-
pared with untreated eyes (range, 84–128lV), with interocular
differences averaging 35% (range, 22–50%). Interocular dif-
ferences in wild-type mice for the cone-isolated ERG had a
mean near zero (range, -35 to +38%) (Fig. 2B). In summary,
interocular differences for all four ERG parameters in rd6 mice
were significantly larger than in wild-type mice. Taken to-
gether with the fact that untreated eyes at this age had re-
duced retinal function compared with wild-type, the results
suggest that the vector–gene injection rescued rod- and cone-
mediated ERG function in the rd6 mice.
Light microscopy was used to evaluate the effect of the
gene therapy on rd6 mice (Fig. 2C). Treated eyes had greater
numbers of photoreceptor nuclei compared with the con-
tralateral untreated eyes. The ONL of untreated rd6 eyes was
about 5–7 nuclei thick (Fig. 2C, middle), whereas the vector-
injected retina had 9 or 10 nuclei (Fig. 2C, right), which is
similar to that in the wild-type eyes (Fig. 2C, left). ONL
thickness (mid-superior retina) was 47.8–4.9lm in treated
retinas compared with 29.9–1.7lm in untreated rd6 retinas
(p<0.05) (Supplementary Fig. S2A). Treated rd6 retinas
also had well-organized, normal length outer segments, in
contrast to the untreated eye, in which outer segments were
both shortened and disorganized. Abnormal cells, likely of
phagocytic nature (Hawes et al., 2000), were visible in the
subretinal space of untreated rd6 mice (Fig. 2C, arrows,
middle) but absent in treated eyes.
MFRP expression after gene therapy in rd6 mice
MFRP expression was evaluated by immunohistochemis-
try in retinal sections (Fig. 3). Previous studies have shown
that MFRP in wild-type mice is predominantly expressed in
the RPE and ciliary epithelium whereas MFRP protein is not
detected in rd6 eyes (Kameya et al., 2002; Mandal et al., 2006a;
Won et al., 2008). This is confirmed in the present study (Fig.
3A and B). After gene therapy, intense labeling was present in
the RPE (Fig. 3C), and it was similar to the pattern of MFRP in
wild-type retina (Fig. 3A). When viewed at low magnification,
the treated rd6 eye displayed widespread and nearly contin-
uous expression of MFRP in the RPE (Fig. 3C, inset). This
indicates that a wide expanse of retina was included in the
subretinal injection. In addition, MFRP expression, as driven
by the ubiquitous smCBA promoter, was detectable in pho-
toreceptor inner segments and ONL when the images were
purposely overexposed to enhance the low-level non-RPE
transgene expression pattern throughout the retina (Supple-
mentary Fig. S2B).
Western blot analysis confirmed the presence of an im-
munoreactive MFRP band in treated eyes only (Fig. 3F). The
MFRP immunoreactive band migrated at about 120kDa, a
much larger size than the predicted molecular mass of
65kDa of wild-type protein, but consistent with results of
previous studies (Won et al., 2008; Fogerty and Besharse,
2011). This high molecular weight band suggests that MFRP
may exist as a dimer in RPE cells. Previous studies have
shown that some of the frizzled proteins are capable of
forming homodimers, and a region containing the cysteine-
rich domain is implicated in this process (Carron et al., 2003).
Cysteine-rich domains have also been shown to form a
conserved dimer interface within crystal structures, sug-
gesting that dimerization may have a biological function in
frizzled receptor signaling (Dann et al., 2001).
In earlier studies MFRP expression was found to be re-
stricted to the base of the RPE apical membrane (Mandal
et al., 2006a), but we observed intense labeling of RPE mi-
crovilli in both wild-type and treated rd6 mice (Fig. 3D and
E). We further confirmed this finding by staining with an
anti-ezrin antibody, a marker of RPE apical processes (Sup-
plementary Fig. S3).
MFRP-RP: A complex phenotype showing
developmental and degenerative abnormalities
Before relatively routine molecular analysis, clinical re-
ports noted the rare association of high hyperopia, micro-
phthalmos, and retinal degeneration with or without other
ocular abnormalities (e.g., Buys and Pavlin, 1999; Ghose
et al., 1985; MacKay et al., 1987). The MFRP gene, first iden-
tified more than a decade ago (Katoh, 2001), was initially
associated only with humans having high hyperopia, re-
duced axial length, but no retinal degeneration (Sundin et al.,
2005). Screening for MFRP mutations in a wide spectrum of
retinal degenerations was negative (Pauer et al., 2005) and
species differences were used to explain the lack of retinal
degeneration in humans (Sundin, 2005). More recently,
however, autosomal recessive families with MFRP mutations
were identified with RP, posterior microphthalmos, foveal
abnormalities, and optic disc drusen (Ayala-Ramirez et al.,
2006; Crespı ´ et al., 2008; Zenteno et al., 2009; Mukhopadhyay
et al., 2010). A list of clinical and molecular features of the 16
published patients (age range, 16–60 years), representing 7
families, is provided (Supplementary Table S1). Visual acu-
ities varied from 20/25 to no light perception (NLP). All had
high hyperopia (range, +13.50 to +29.00) and abnormal
ERGs. Corneal diameters were normal. Data from the MFRP-
RP patient in the present study are consistent with the
clinical phenotype in these other reports (Supplementary
The convexity forming a domelike configuration in the
central retina with persistent inner retinal structure across
the presumptive fovea in human MFRP disease suggests a
disturbance in the complex early cell migrations that lead to
normal foveal development. Nine of the previously pub-
lished patients with MFRP-RP had OCT scans and all
showed the lack of a foveal pit and thickening of inner retinal
tissue across the foveal region (Ayala-Ramirez et al., 2006;
Crespı ´ et al., 2008; Zenteno et al., 2009; Mukhopadhyay et al.,
2010). We postulate that this is the result of foveal mal-
development (Sundin et al., 2008), likely complicated by ef-
fects of degenerative retinal disease. Formation of the normal
foveal pit occurs during the second half of gestation and is
due to progressive thinning of ganglion cell and inner
OCULAR GENE THERAPY FOR MFRP-RP373
nuclear cell layers that overlie the foveal cone cells due to
their centrifugal displacement (reviewed in Provis et al.,
1998). The process is complete at about 1 year of life. Another
process, but a centripetal one, leads to increasing cone cell
density in the foveal pit and this continues for years after
birth. Taken together, the good visual acuity in many pa-
tients with MFRP-RP (Supplementary Table S1), normal in-
ner and outer segment thickness measurements in our
patient, and thicker than normal foveal ONL suggest that the
cone cell centripetal movement has occurred. It is also con-
ceivable that Muller cell swelling secondary to noncystic
macular edema is the basis of the thickened hyporeflective
layer (Jacobson et al., 2006). The excessively thickened su-
perficial hyperreflective layer above the cone cells suggests
persistence of tissue that should have moved centrifugally if
there was normal development. A comparison with different
retinopathies indicated that even the thickest of other pre–
cone cell layers we examined are far thinner than the MFRP-
RP layer. The OCT appearance without a foveal depression
has some similarities but also differences from other mal-
development retinopathies such as albinism (Marmor et al.,
2008; McAllister et al., 2010) and nanophthalmic eyes without
retinal degeneration (Bijlsma et al., 2008). The findings in the
extracentral retina of photoreceptor degeneration and inner
retinal thickening point to retinal remodeling in MFRP-RP,
as we have noted in many retinal degenerative diseases (e.g.,
Jacobson et al., 2006; Aleman et al., 2009; Cideciyan et al.,
Gene therapy in the rd6 mouse model
of MFRP disease
The AAV8 (Y733F) tyrosine capsid mutant has emerged as
a promising vector for the treatment of rapidly degenerating
animal models of RP caused by mutations in photoreceptor
cells (Pang et al., 2011). Here, we demonstrate the ability of a
subretinally delivered AAV8 (Y733F) vector containing a
smCBA promoter driving expression of the murine Mfrp
gene to rescue retinal function and prevent photoreceptor
cell death in the rd6 mouse, the early-onset RPE-based model
of MFRP-RP. To minimize the surgical trauma associated
with subretinal injections and to still be able to provide rapid
and widespread transgene expression before the initiation of
cell death, treatment was provided on P14 in rd6 mice, co-
incident with eye opening when the photoreceptor cell layer
was still fully intact. AAV8 (Y733F)-smCBA-MFRP vector
delivery at this age led to significantly higher ERG ampli-
tudes relative to untreated eyes when assessed 6 weeks later.
Histology of treated retinas revealed preserved photorecep-
tor ONL with better organized inner and outer segments
than control untreated rd6 retinas. Moreover, accumulation
of abnormal, phagocytic-like cells in the subretinal space was
not observed in vector-treated eyes. Treatment also led to
intense and widespread expression of the MFRP transgene,
predominantly localized to the apical RPE membrane and its
microvilli, where the wild-type protein normally resides in
The function of the MFRP protein is not clear at this time,
and the mechanism of photoreceptor degeneration in rd6
mice could be multifactorial. The presence of MFRP
throughout the apical membrane and its actin-rich micro-
villi, both in wild-type and treated rd6 mice, suggests that it
may play a structural role by maintaining the normal
morphology of the RPE apical processes. MFRP expression
normally increases markedly after birth, coincident with the
development of microvilli and outer segments in mice (Won
et al., 2008). Thus, rd6 retinal degeneration may be caused
by compromised RPE microvilli leading to adhesion defects
between RPE and photoreceptor outer segments. In addi-
tion, experiments suggest that MFRP and CTRP5 physically
interact, and could therefore be both functionally and
transcriptionally dicistronic (Mandal et al., 2006a; Shu et al.,
2006). CTRP5 contains a short-chain collagen sequence and
a C-terminal globular complement 1q (C1q) domain that
appears to bind the extracellular MFRP region containing
tandem repeats of two cubilin (CUB) domains and a low-
density lipoprotein receptor-related sequence (Shu et al.,
2006). C1q is a key component of the classical pathway of
complement activation, and is known to be involved in
many critical processes including innate and adaptive im-
munity, inflammation, apoptosis,
monocyte chemotaxis (Kishore et al., 2004; Lu et al., 2008).
Interestingly, a study describing a murine Mfrp null mutant
has shown that CTRP5 is upregulated in both rd6 and
Mfrp174delG(Fogerty and Besharse, 2011). Thus, the lack of
MFRP protein might lead to uncontrolled signaling by
CTRP5, triggering the migration of phagocytic cells from
the choroid into the subretinal space, as seen in rd6 mice.
Morphological characterization of an Mfrp/Ctrp5 double-
knockout mouse could prove useful for testing this hy-
pothesis in future studies.
Realistic potential for gene-based therapy in MFRP-RP
Is MFRP-RP a target for future gene therapy trials? Al-
though a limited number of patients have been reported to
date, there is a recognizable human phenotype with a dra-
matic refractive error that should make clinical and genetic
screening more productive in the future than previously
(Pauer et al., 2005). Evidence of central retinal maldevelop-
ment and early peripheral retinal degeneration with loss of
normal retinal architecture indicating remodeling makes a
complex target for gene therapy as it is currently performed.
Despite reports of safety and efficacy of subretinal gene
therapy as currently performed in the RPE65 form of LCA
(reviewed in Cideciyan, 2010), a surgically induced subfoveal
retinal detachment in a well-functioning but maldeveloped
central retina may present more risk than benefit. The patient
with MFRP-RP whom we examined, however, had preserved
although abnormal peripheral retinal function (at the end of
the second decade of life) by ERG and psychophysics, both for
rod and cone photoreceptor systems. This suggests the non-
foveal retina is a potential therapeutic target. In terms of rel-
evant rd6 experiments to perform, our relatively short-term
and preliminary studies with one vector serotype demand to
be confirmed and extended to longer term efficacy and safety
studies, dose–response experiments, and future work to ad-
dress the potentially safer approach of intravitreal delivery of
Mfrp cDNA driven by RPE cell-specific promoters (e.g., ty-
rosine capsid mutant AAV vectors; Petrs-Silva et al., 2011).
Supported in part by NIH grants R01EY11123 and
P30EY021721, and by grants from the Macula Vision
374 DINCULESCU ET AL.
Research Foundation, Foundation Fighting Blindness, and
Research to Prevent Blindness. A.V.C. is an RPB Senior Sci-
Author Disclosure Statement
W.W.H. and the University of Florida have a financial in-
terest in the use of AAV therapies, and own equity in a
company (AGTC Inc.) that might, in the future, commercialize
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Address correspondence to:
Dr. Samuel G. Jacobson
Scheie Eye Institute, Department of Ophthalmology
Perelman School of Medicine
University of Pennsylvania
Philadelphia, PA 19104
Dr. William W. Hauswirth
Department of Ophthalmology
University of Florida
Gainesville, FL 32610
Received for publication September 9, 2011;
accepted after revision December 4, 2011.
Published online: December 5, 2011.
376 DINCULESCU ET AL.