Session 414 AMD Post-VEGF Novel Therapies

Transcription

ARVO 2016 Annual Meeting Abstracts
414 AMD Post-VEGF Novel Therapies
Wednesday, May 04, 2016 8:30 AM–10:15 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 4418–4447/A0323–A0352
Organizing Section: Retina
Program Number: 4418 Poster Board Number: A0323
Presentation Time: 8:30 AM–10:15 AM
Assessment of Retinal Pigment Epithelium (RPE) Atrophy in a
Phase 2b Study of a Platelet Derived Growth Factor inhibitor
(Fovista®), in combination with a Vascular Endothelial Growth
Factor inhibitor (Ranibizumab) for Neovascular Age-Related
Macular Degeneration (NAMD)
Thomas A. Ciulla1, 2, Glenn J. Jaffe3, Samir Patel1. 1Ophthotech, New
York, NY; 2Ophthalmology, Indiana University School of Medicine,
Indianapolis, IN; 3Ophthalmology, Duke University School of
Medicine, Durham, NC.
Purpose: In several large NAMD randomized controlled trials
(RCT), there is an increased risk of developing new geographic
atrophy with monthly compared to discontinuous intravitreal vascular
endothelial growth factor (VEGF) inhibition. Herein, we describe
the occurrence of RPE atrophy over 24 weeks in the Phase 2b Study
of Fovista®, a platelet derived growth factor (PDGF) inhibitor in
combination with a VEGF inhibitor for NAMD.
Methods: 449 patients with NAMD were randomized in a
prospective, controlled, superiority trial to receive one of the
following treatment regimens every 4 weeks for 24 weeks:
Fovista® 0.3 mg with ranibizumab 0.5 mg; Fovista® 1.5 mg with
ranibizumab 0.5 mg; or sham with ranibizumab 0.5 mg. OCT, fundus
photographic, and fluorescein angiographic images were evaluated
by readers masked to treatment assignment at a centralized reading
center.
Results: The combination of Fovista® (1.5 mg) with ranibizumab
met the pre-specified primary endpoint of mean visual acuity gain
superiority compared to ranibizumab monotherapy (10.6 ETDRS
letters at week 24, compared to 6.5 letters, p=0.019). On OCT,
fundus photography and fluorescein angiography eyes that received
Fovista®1.5 mg combination therapy had greater inhibition of
choroidal neovascularization (CNV) and fibrosis compared to those
receiving ranibizumab monotherapy. At 24 weeks, RPE atrophy was
evident in 20.8% (30/144) eyes in the ranibizumab monotherapy
group, 16.8% (24/143) in the Fovista® 0.3 mg combination therapy
group, and 15.9% (23/145) in the Fovista® 1.5 mg combination
therapy group.
Conclusions: At 24 weeks, compared to ranibizumab monotherapy,
Fovista® combination therapy resulted in reduction of sub-retinal
fibrosis and CNV growth, while also showing a lower occurrence
of RPE atrophy. Fovista® combination therapy may address the
underlying mechanisms of chronic insidious vision loss in NAMD.
Commercial Relationships: Thomas A. Ciulla; Glenn J. Jaffe,
Ophthotech (C); Samir Patel, Ophthotech, Ophthotech (I)
Clinical Trial: NCT01089517
CNV lesion characteristics as a predictor of visual outcome in
wet AMD patients receiving combination therapy of intravitreal
anti-VEGF therapy and topical Squalamine lactate ophthalmic
solution
David M. Brown1, Avner Ingerman2, Shawn P. Shearn2,
Jason S. Slakter3. 1Retina Consultants of Houston, Houston, TX;
2
Ohr Pharmaceutical, Inc, New York, NY; 3Vitreous Retina Macula
Consultants of New York, New York, NY.
Purpose: Neovascular age-related macular degeneration (AMD)
is currently treated by intravitreal (IVT) injections of anti-VEGF
agents. Multiple, frequent anti-VEGF IVT injections are required,
and improvement in visual function is often sub-optimal. Squalamine
lactate 0.2% solution is administered topically, and functions by
blocking intracellular signaling pathways common to VEGF, PDGF,
and bFGF. An exploratory Phase II study examined the safety and
efficacy of treatment with topical Squalamine, administered BID for
9 months, in combination with PRN (as-needed) IVT ranibizumab
injections.
Methods: 142 patients enrolled in a phase II, double masked, placebo
controlled study. All patients received a single IVT injection of
ranibizumab at baseline, followed by criteria-based PRN ranibizumab
IVT injections monthly to month 9. Patients were randomized
1:1 to receive either placebo eye drops or Squalamine eye drops
BID to month nine. 128 patients (63 in the placebo/Ranibizumab
monotherapy group and 65 patients in the Squalamine/
combination group) completed the month 9 endpoint. Patients
with total lesion area ≤ 12 disc areas, choroidal neovascularization
(CNV) affecting > 50% of the total lesion area, and all angiographic
lesion types (occult and classic) were included in the study. Best
Corrected Visual Acuity (BCVA) was assessed monthly and
compared to baseline. An analysis of visual outcome as a function of
lesion characteristics (presence and size of classic and occult CNV)
was performed.
Results: At month 9, the mean BCVA gain from baseline was 7.8
letters for all patients in the Combination group (N=65), compared
to 5.3 letters for all patients in the ranibizumab monotherapy group
(N=63) (P=0.25). An analysis of visual acuity outcome as a function
of the occult CNV size demonstrated a strong correlation in the
combination therapy group (p=<0.0001) but not with ranibizumab
monotherapy (Table).
Conclusions: Angiographic CNV lesion characteristics were
predictive of visual acuity outcomes in combination therapy. Baseline
occult CNV area shows the highest correlation to visual outcome in
combination treatment with IVT anti-VEGF and topical Squalamine.
Patients with occult CNV <10 mm2 represent an optimal target
population for combination therapy in clinical trials.
Commercial Relationships: David M. Brown, Santen (F),
Avalanche (C), Regeneron (F), Bayer (C), Santen (C), Allergan
(F), Alcon (C), Genentech (C), Alcon (F), Alimera (C), Regeneron
(C), Alimera (F), Genentech (F), Allergan (C); Avner Ingerman,
Ohr Pharmaceutical, Inc. (I), GSK (C), Ohr Pharmaceutical,
Inc, Neurotech (C); Shawn P. Shearn, Ohr Pharmaceutical,
Inc (I), Ohr Pharmaceutical, Inc; Jason S. Slakter, Santen (F),
Lpath (F), Regeneron (F), GSK (F), Roche (F), Allergan (F), Ohr
Pharmaceutical, Inc (I), Sanofi (F), Tyrogenix (F), Tyrogenix
(C), Regeneron (C), Ohr Pharmaceutical, Inc, Genentech (F),
Thrombogenics (F), Bayer (F)
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
Treatment of mature neovascularisation by targeting
interleukin-8
Peter Heiduschka, Lisa Pohlmann, Daniel Niekämper, Nicole Eter.
Opthalmalogy, University of Muenster Medical School, Muenster,
Germany.
Purpose: As there can be a limited treatment success by anti-VEGF
drugs in patients with neovascular diseases, e.g. if neovascularisation
is maturated, we are looking for other factors that could play a role
in CNV in the experimental model of laser-induced CNV in mice.
We found a strong immunoreactivity for interleukin-8 (IL-8) after
laser treatment, and we checked whether treatment with an anti-IL-8
antibody has an effect in the laser-induced CNV model.
Methods: Eyes of C57BL/6J mice were treated with an argon laser
to induce CNV. Two weeks after laser treatment, antibodies against
the following factors were injected intravitreally in groups 1 to 7:
PBS as a control, VEGF, IL-8, VEGF+IL-8, PDGF-beta, PDGFbeta+IL-8, PDGF-beta+VEGF (n=8 for each group). Size of CNV
was evaluated in vivo by scanning laser ophthalmoscopy (SLO)
and fluorescein angiography (FA) two weeks after laser treatment,
i.e. before intravitreal injections, and two weeks later. Afterwards,
laser spots were labelled with isolectin B4 in choroidal flat mounts.
We evaluated the changes of laser spot sizes as obtained by in vivo
imaging by SLO (proliferation area) and FA (fluorescein leakage)
expressed as ratio between the values before intravitreal injection and
two weeks afterwards. Laser spot sizes in choroidal flat mounts were
determined in mm2. Statistical analysis was performed by the oneway ANOVA test with Tukey correction.
Results: For groups 1 to 7, mean ratios of changes of the
proliferation area were 1.23, 1.05, 0.95, 0.88, 1.16, 0.85 and 1.69,
mean ratios of changes of fluorescein leakage sizes were 0.97, 0.81,
0.81, 0.82, 2.10, 1.44 and 0.51, and mean laser spot sizes in choroidal
flat mounts were 0.029, 0.019, 0.018, 0.014, 0.024, 0.016 and 0.020
mm2, respectively. CNV parameters were smaller when anti-VEGF or
anti-IL-8 were injected alone, combined or together with anti-PDGFbeta, reaching statistical significance for the values of proliferation
area and laser spot size in choroidal flat mounts after injection of
anti-VEGF + anti-IL-8. No effects were observed when anti-PDGF
was injected alone.
Conclusions: The results clearly indicate that sizes of mature
laser spots in the experimental model of laser-induced CNV are
reduced after intravitreal injection of an anti-IL-8 antibody alone
or in combination with anti-VEGF or anti-PDGF-beta antibodies,
thus possibly opening a new way to deal with treatment-resistive
neovascularisations.
Commercial Relationships: Peter Heiduschka, Novartis (F);
Lisa Pohlmann, None; Daniel Niekämper, None; Nicole Eter,
None
SEMAPHORIN-3E RESISTANT TO CLEAVAGE BY FURINS
INHIBITS CHOROIDAL NEOVASCULARIZATION
Agustina C. Palacio1, Shira Toledano2, Huayi Lu1, Ofra Kessler2,
Yoreh Barak3, Gera Neufeld2, Shlomit Schaal1. 1Ophthalmology,
University of Louisville, Louisville, KY; 2Cancer Research and
vascular Biology Center, The Bruce Rappaport Faculty of Medicine,
Technion, Israel Institute of Technology, Haifa, Israel; 3Department of
Ophthalmology, Rambam Medical Center, Haifa, Israel.
Purpose: The exudative form of age-related macular degeneration
(AMD) results in choroidal neovascularization (CNV). Anti-VEGF
agents are widely used to treat AMD, however the high rate of non-
responders calls for the exploration of anti-angiogenic agents that
act independently from the VEGF pathway. This study was designed
to investigate whether a point mutated form of the anti-angiogenic
semaphorin-3E which was rendered resistant to cleavage by furin like
pro-protein convertases (UNCL Sema3E) can be used to effectively
inhibit laser induced CNV formation in a rat and a mouse model.
Methods: CNV was induced in the eyes of Evans-Long rats (n=16)
and also in the eyes of C57B mice (n=20) by laser photocoagulation
followed by an intravitreal injection of either 50 µg or 5 µg for the
rat and the mice model respectively UNCL-Sema3E, 50 µg Avastin®
(bevacizumab), 5 µg EYLEA® (aflibercept) or vehicle as control.
After a week flat whole mounts of retinas where used to determine
CNV frequency and size. Results were assessed by the staining of
blood vessels with isolectin or with dextran green and calculating
the area of stained blood vessels using the Image-J morphometric
software. Visual function was assessed using a non-invasive
OptoMotry © optokinetic testing system.
Results: UNCL-Sema3E (50 µg) injected into the vitreous cavity of
rats reduced the area of laser induced CNV by 50% (64040 ± 7321
μm2 for controls (n=61) vs 32720 ±- 2369 μm2 (n=65), P<0.001)
displaying efficacy similar to that of bevacizumab (32062±- 1806μm2
(n=54), P<0.001). UNCL-Sema3E (5µg) injected into the vitreous
cavity of mice reduced the area of laser induced CNV by 35%
(121063± 16957μm2 for controls (n=48) vs 78400± 8802μm2 (n=64),
P<0.05) displaying somewhat lower efficacy than that of EYLEA
(48713± 7255 μm2 (n=40), P<0.0001).
Conclusions: UNCL-Sema3E significantly inhibits laser induced
CNV formation with similar efficiently as bevacizumab, but less
efficiently than EYLEA. UNCL-Sema3E did not compromise visual
acuity in optokinetic assays. UNCL-Sema3E mechanism of action
differs from current therapies that block VEGF, therefore, UNCLSema3E carries the promise to become an adjunct therapeutic agent
for the treatment of exudative AMD that is refractory to current
treatments.
Commercial Relationships: Agustina C. Palacio, None;
Shira Toledano, None; Huayi Lu, None; Ofra Kessler, None;
Yoreh Barak, None; Gera Neufeld, None; Shlomit Schaal, None
Support: This project is supported by the Binational Science
Foundation (Schaal, Barak 2015), by an unrestricted institutional
grant from Research to Prevent Blindness (RPB), and by a grant from
the Israel Science Foundation (Neufeld).
Cell Recruitment and Molecular Changes to the Retina following
Sub-threshold Micropulse Retinal Phototherapy
Sergio Caballero1, David L. Kent2, Maria B. Grant3. 1Pharmacology/
Therapeutics, University of Florida, Gainesville, FL; 2The Vision
Clinic, Kilkenny, Ireland; 3Ophthalmology, Indiana University Purdue
University Indianapolis, Indianapolis, IN.
Purpose: Sub threshold retinal phototherapy (SRP) has been shown
to be clinically efficacious for the treatment of retinal disease
including diabetic macular edema without visible signs of retinal
damage. SRP delivers short impulse light energy absorbed by the
retinal pigment epithelium (RPE) only, sparing the neurosensory
retina and achieving photo stimulation rather than photocoagulation.
We investigated the use of SRP stimulation of the RPE and choroid to
recruit hematopoietic stem cells (HSC) to the RPE.
Methods: GFP chimeric mice were generated by bone marrow
ablation through irradiation of female C57Bl/6J and reconstituting
with bone marrow cells from C57BL/6-Tg(ACTB-EGFP) donor
males. 4 months later, mice were subjected to SRP of the retina in one
eye using an infrared laser (Iridex OcuLight SLx MicroPulse 810nm)
coupled to a slit lamp. Variable duty cycles of 5, 10, 15 and 20% (n=5
animals each duty cycle) for 0.1sec at 250mW and a spot size set to
50µm were used to establish optimal conditions. 20-30 applications
were administered to each eye circumferentially placed between
50-100 µm from the optic disc. Another cohort of 5 animals did not
receive laser application and was used as negative control. mRNA
expression for hsp70 and 90, crystalline, HIF-1α, VEGF, CXCL-12,
and CXCR-4 in both the neurosensory retina and the posterior cup
was determined. Retinas were processed for immunohistochemistry
for GFP.
Results: No visible laser burn or scar was noted. GFP+ cells migrated
to the RPE layer in a duty cycle-dependent fashion. hsp70 mRNA
peaked at 2h post-laser in the neural retina and at 4h in the posterior
cup. mRNA for hsp90A dramatically peaked in both the neural
retina and the posterior cup at 2h. mRNA for CXCL12 and its
receptor CXCR4 in the posterior cup occurred 2h post laser injury,
and increased further by 4h. HIF-α mRNA was reduced at 2h, but
increased at 4h; however, only in the posterior cup. VEGF mRNA
increased at 4-6h post laser.
Conclusions: Bone marrow-derived cells are recruited to the retina
including the RPE layer following sub threshold laser. The subthreshold laser likely stimulates the extracellular environment of the
RPE-photoreceptor layers resulting in release of hsp70, hsp90 and
crystalline induction, which is then followed by cytokine release
(CXCL12 and VEGF) that sustain the tissue signals to recruit bone
marrow cells to the retina.
Commercial Relationships: Sergio Caballero, None;
David L. Kent, None; Maria B. Grant, None
Support: EY007739, EY023629
Semaphorin-3C Resistant To Cleavage By Furins Inhibits
Choroidal Neovascularization
Shira Toledano1, Huayi Lu2, Agustina Palacio2, Ofra Kessler1,
Shlomit Schaal2, Gera Neufeld1, Yoreh Barak3. 1Cancer Research and
vascular Biology Center, The Bruce Rappaport Faculty of Medicine,
Technion - Israel Institute of Technology, Haifa, Israel; 2Department
of Ophthalmology and Visual Sciences, University of Louisville
School of Medicine, Louisville, KY; 3Department of Ophthalmology,
Rambam Medical Center, Haifa, Israel.
Purpose: Choroidal neovascularization (CNV) is a major blinding
consequence of several retinal diseases including the exudative
form of age-related macular degeneration (AMD). Anti-VEGF
agents are widely used to treat AMD, however the high rate of nonresponders calls for the exploration of anti-angiogenic agents that
act independently from the VEGF pathway. This study was designed
to investigate whether a point mutated form of the anti-angiogenic
semaphorin-3C which was rendered resistant to cleavage by furin
like pro-protein convertases (FR-Sema3C) can be used to effectively
inhibit laser induced CNV formation in a mouse model.
Methods: CNV was induced in the eyes of C57B mice (n=15) by
laser photocoagulation followed by an intravitreal injection of either
100 ng FR-Sema3C, 5µg EYLEA® (aflibercept) or vehicle as a
control. After a week flat whole mounts of retinas where used to
determine CNV development and size. Results were assessed by the
staining of blood vessels with dextran green and by calculation of the
area of stained blood vessels using Image-J morphometric software.
The effects of FR-sema3C injection on the visual function of healthy
mice were assessed using a non-invasive OptoMotry© optokinetic
testing system.
Results: FR-Sema3C (100 ng) injected into the vitreous cavity of
mice reduced the area of laser induced CNV by 44% as compared to
controls (121063± 16957μm2 for controls (n=48) vs 53383±5779μm2
(n=40), P<0.001). This efficacy was similar to that of EYLEA
(40%, 48713± 7255μm2 (n=40), P<0.0001). The optokinetic essays
indicated that at this dose FR-sema3C does not compromise visual
acuity. FR-Sema3C also inhibited significantly laser induced CNV
using lower doses (10 and 50 ng per injection) although the inhibition
was less effective than at 100 ng per injection (142578± 23886μm2
for controls (n=16) vs 100 ng (53153± 10094μm2 (n=24), P<0.001)
,50 ng (93836± 12890μm2 (n=24), P<0.05) and 10 ng (95042±
16348μm2 (n=24), P<0.05).
Conclusions: FR-Sema3C inhibits laser induced CNV formation in
the mouse model as efficiently as EYLEA and seems to be devoid
of toxic effects. FR-sema3C mechanism of action is independent
of VEGF. This suggests that FR-Sema3C may be considered as a
possible therapeutic agent for the treatment of exudative AMD that is
refractory to current VEGF targeting agents.
Commercial Relationships: Shira Toledano, None; Huayi Lu,
None; Agustina Palacio, None; Ofra Kessler, None;
Shlomit Schaal, None; Gera Neufeld, None; Yoreh Barak, None
Support: This project is supported by the Binational Science
Foundation (Schaal, Barak 2015), by an unrestricted institutional
grant from Research to Prevent Blindness (RPB), and by a grant from
the Israel Science Foundation (Neufeld).
Laser Thermal Stimulation of the Retina (TS-R) reduces
Thickness of Bruch’s Membrane (BrM) in Apolipoprotein (Apo)
E knock out Mice
Jan Tode1, Elisabeth Richert1, Alexa K. Klettner1, Ralf Brinkmann2,
Stefan Koinzer1, Johann Roider1. 1Department of Ophthalmology,
Christian-Albrechts-University, University Medical Center, Kiel,
Germany; 2Medical Laser Center Lübeck, Lübeck, Germany.
Purpose: An increase in BrM thickness is one of the main features
of non exsudative age related macular degeneration (dry AMD).
Currently there is no adequate treatment for dry AMD. We investigate
the effect of TS-R in an AMD mouse model.
Methods: Ten eyes of five 4 month old ApoE knock out mice
(B6.129P2-Apoetm1Unc/J) were used. We treated 5 eyes of 5 ApoE
knock out mice with a TS-R laser (laser parameters: 532 nm
wavelength, continuous wave, 50 µm spotsize, 10 ms duration, mean
power 5.4 mW) and used the fellow eye as control. We applied 70 to
100 laser spots distributed uniformly with a spacing of 2 spots. Power
was chosen 70 % below clinical visibility.
Eyes were examined by ophthalmoscopy, optical coherence
tomography (OCT) and fluorescein angiography (FLA) at the day of
treatment, 1 day and 1 month after treatment. We enucleated the eyes
after 1 month and analyzed BrM thickness by transmission electron
microscopy (TEM) in a blinded standardized manner.
Results: Fundus images revealed that all ApoE knock out mice had
AMD associated alterations like drusen and areas of hypopigmented
retinal pigment epithelium (RPE). Drusen were also visible in OCT.
FLA did not reveal any pathological findings.
Subthreshold TS-R laser spots were not visible by fundus imaging,
OCT and FLA, 2 hours, 1 day or 1 month after laser treatment.
There was no significant change in the number of drusen of the
treated eye before TS-R (mean 4, +/- 4) compared to the treated
eye after TS-R (mean 7, +/- 3) and compared to the control eyes
(mean 5, +/- 2).
However, TEM revealed a significant reduction (p< 0.001) of BrM
thickness in laser treated eyes (mean 524.8 nm, SEM 11.4 nm)
compared to their fellow control eyes (mean 585.7 nm, SEM 12.9
nm). BrM of all treated eyes was thinner than BrM of their fellow
control eyes.
Conclusions: Laser thermal stimulation of the retina TS-R reduces
BrM thickness in ApoE knock out mice without damage to the retina.
It may be a treatment option for dry AMD.
Commercial Relationships: Jan Tode, None; Elisabeth Richert,
None; Alexa K. Klettner, None; Ralf Brinkmann, None;
Stefan Koinzer, None; Johann Roider, None
Support: BMBF grant 13GW0043D
Clinical evaluation of stereotactic low-voltage X-ray irradiation
for neovascular age related macular degeneration
Maximilian Kurz, Annekatrin Holzhey, Salvatore Grisanti,
Mahdy Ranjbar. Ophthalmology, University of Lübeck, Lübeck,
Germany.
Purpose: To analyze the clinical and morphological outcome of
stereotactic low voltage X-ray irradiation (SRT) in patients with
neovascular age-related macular degeneration (nAMD) under reallife circumstances.
Methods: 37patients, selected by the criteria for best responders as
determined by the INTREPID study, were treated with 16 Gray SRT.
All patients received pro re nata (PRN) aflibercept, bevacizumab
or ranibizumab. Evaluation based on visual acuity, morphological
changes as described by OCT, multifocal-ERG and number of
applied injections.
Results: Patients showed a significant increase of visual acuity,
morphological improvement of retinal integrity, despite significantly
less injections. No electrophysiological adverse effects of SRT were
recorded during the first year after treatment.
Conclusions: Our results might indicate that a single dose of
low-voltage x-ray irradiation in patients with age-related macular
degeneration significantly reduces intravitreal injections over 12
months. This is not only relevant for the patients from a quality of
life perspective, but also due to the demographic trend of our society
from an economical point of view.
Commercial Relationships: Maximilian Kurz, None;
Annekatrin Holzhey, None; Salvatore Grisanti, None;
Mahdy Ranjbar, None
A comparison of expected dwell times and dose variations for
neovascular age-related macular degeneration (n-AMD) patients
treated with an episcleral brachytherapy device
Kamaljit S. Balaggan5, 6, Russell J. Hamiliton1, Mary E. Drew2,
Praveen J. Patel4, Daren Hanumunthadu4, Reid Schindler3,
Marie Restori5, Tomas Ilgins4, Meghan McGovern2,
Laurence Marsteller2. 1Radiation Oncology, University of Arizona,
Tucson, AZ; 2Salutaris Medical Devices, Inc., Tucson, AZ; 3Retina
Specialists of Southern Arizona, Tucson, AZ; 4NIHR Biomedical
Research Centre at Moorfields and UCL, London, United
Kingdom; 5Moorfields Eye Hospital, London, United Kingdom;
6
Ophthalmology, Wolverhampton Eye Infirmary, Wolverhampton,
United Kingdom.
Purpose: To evaluate variations in dwell times and doses expected
when using an episcleral brachytherapy device for treatment of
n-AMD based on Optical Coherence Tomography and Ocular
Ultrasound imaging modalities. To determine if a mean target depth
is appropriate for all patients receiving episcleral brachytherapy.
Methods: Data from 40 eyes from 40 subjects with known n-AMD
was acquired through the Distance of Choroid Study (DOCS)
conducted at Moorfields Eye Hospital. The purpose of DOCS was
to determine the repeatability and limits of agreement of existing
imaging modalities for the determination of retinal, choroidal and
scleral thickness at the fovea and choroidal neovascularization (CNV)
lesion in vivo. Each subject underwent EDI SD-OCT, SS-OCT and
Ocular Ultrasound (O-US). Using the percentage depth dose for a Sr90 episcleral brachytherapy device, dwell times and doses delivered
are computed to determine the expected variations of each subject.
Results: The mean distance to target depth and the 95% confidence
interval (CI) determined by combining O-US with SD-OCT were
1326 (956,1696)mm and with SS-OCT were 1332 (970,1693)mm.
The calculated corresponding mean dwell times and 95% (CI) were
334 (223, 445)s and 335 (226, 445)s for SD-OCT and SS-OCT
determined depths, respectively. The patient population average target
depth for treatment planning results in dose variations of +/-33% over
the 95% CI.
Conclusions: The mean dwell times showed no significant
difference between SD-OCT and SS-OCT. Each patient should have
individualized imaging studies to determine the target depth.
The apex dose for each patient found using the dwell time calculated
based on the population average depth. The solid line is the intended
prescription dose of 24Gy and the dotted lines are ±15% variations
above and below this. The apex dose differs by more than 15% for
40% (16/40) of the patients if the population average depth is used
for the dwell time calculation.
Depth dose curve along a line through the center of the applicator
and perpendicular to it. The dose changes rapidly with depth. When
the prescription dose (24Gy) is delivered at a depth equal to the
mean apex depth of the patients in our study (1.326mm), the doses at
the apex depths within the 95% confidence interval of our patients,
0.956mm to 1.696mm, range from 33.6Gy to 17.1Gy.
Commercial Relationships: Kamaljit S. Balaggan, DORC (R),
SalutarisMD (C), Novartis (R); Russell J. Hamiliton, Salutaris
Medical Devices, Inc. (P), Salutaris Medical Devices, Inc. (I);
Mary E. Drew, Salutaris Medical Devices, Inc.; Praveen J. Patel,
Salutaris Medical Devices Inc (R), Novartis (C), Thrombogenics
NV (C), Heidelberg Inc (F), Topcon Inc (F), Thrombogenics NV (F),
Bayer (C); Daren Hanumunthadu, Salutaris Medical Devices, Inc.
(R); Reid Schindler, Salutaris Medical Devices, Inc. (I), Salutaris
Medical Devices, Inc. (F); Marie Restori, None; Tomas Ilgins;
Meghan McGovern, Salutaris Medical Devices, Inc. (C);
Laurence Marsteller, Salutaris Medical Devices, Inc. (P), Salutaris
Medical Devices, Inc. (S), Salutaris Medical Devices, Inc., Salutaris
Medical Devices, Inc. (I)
Two- year results of a randomized prospective sham-controlled
study comparing proton beam irradiation combined with
ranibizumab with ranibizumab monotherapy for exudative agerelated macular degeneration
Senad Osmanovic1, Elad Moisseiev1, Kavita Mishra2,
Inder Daftari2, Lawrence S. Morse1, Ala Moshiri1, Susanna S. Park1.
1
Ophthalmology, University of California Davis, SACRAMENTO,
CA; 2Radiation Oncology, University of California San Francisco,
San Francisco, CA.
Purpose: Exudative AMD (eAMD) remains a leading cause of severe
visual loss in the developed world, and the need for new treatment
strategies remains strong. We seek to evaluate the efficacy and safety
of combination low-dose proton beam therapy (PBT) with anti-VEGF
versus anti-VEGF monotherapy in exudative AMD, and provide here
an interim 2 year analysis of a prospective randomized control trial.
Methods: 30 Eyes with nvAMD (classic and occult lesions) were
randomized to either sham irradiation or to either 16 or 24Gy PBT,
given in 2 fractions 24 hours apart. Patients underwent monthly
examinations with ranibizumab administered prn. Main outcome
measures were change in BCVA, mean number of injections, severe
visual loss (> 15 letters) and development of radiation retinopathy
or neuropathy at 24 months. Changes in the morphologic features
of neovascular lesions and macular fluid on OCT, fundus and
angiography imaging over were also assessed.
Results: Groups were evenly distributed among 3 study groups
at baseline with no differences in baseline BCVA, lesion size or
demographic profile. Average patient age was 77.7 (SD 7.4) with
baseline visual acuity of 0.61 logMAR (±0.29). Among 18 eyes
completing 24 month follow-up (sham n=5, 16Gy n=6, 24Gy n=7),
BCVA remained stable in all 3 groups during the second year of
study, without significant difference among groups. Mean BCVA at 2
years was 0.59 logMAR ±0.33. The mean number of injections was
statistically lower in both the 16Gy (n=7.4) the 24Gy group (n=5.4)
when compared to sham treatment group (n=11), p= 0.023, p=0.001
respectively. OCT analysis showed a trend towards more reduction
in central macular thickness at 2 years with radiation and anti-VEGF
combined treatment (both 16Gy and 24Gy) when compared to antiVEGF monotherapy. There was no case of severe visual loss. There
were 2 cases of suspected visually insignificant radiation retinopathy,
one for each of the radiation dose subgroups.
Conclusions: Interim analysis at 2-years show no significant safety
concerns combining proton beam irradiation with anti-VEGF therapy
for eAMD. A synergistic effect resulting in less need for re-injection
with anti-VEGF was noted using 16 or 24Gy proton. The complete
2 year follow-up data will be presented to determine whether these
interim findings are sustained.
Commercial Relationships: Senad Osmanovic, None;
Elad Moisseiev, None; Kavita Mishra, None; Inder Daftari, None;
Lawrence S. Morse, None; Ala Moshiri, None; Susanna S. Park,
None
The 5HT1a Agonist Xaliproden Exhibits Anti-oxidant and Antiinflammatory Properties and Protects the Retina in a Mouse
Model of Geographic Atrophy
Alfred S. Lewin, Chulbul M. Ahmed, Manas Ranjan Biswal, Hong Li,
Pingyang Han. Molecular Genetics and Microbiology, University of
Florida, Gainesville, FL.
Purpose: Chronic oxidative stress with concomitant inflammation
have been implicated in the pathogenesis of age related macular
degeneration (AMD). The purpose of this study was to test the
serotonin agonist, xaliproden for protection against geographic
atrophy in a mouse model. This orally available compound has been
tested in clinical trials for other indications.
Methods: ARPE-19 cells were treated with paraquat, an oxidative
stressor with or without xaliproden. In a separate experiment, TNFα was added to the cells in the presence or absence of the drug.
Synthesis of protective antioxidant factors and of inflammatory
cytokines was assayed by RT-qPCR. Survival of cells was measured
by MTS assay and microscopy. Integrity of the cell monolayer
was assessed by immunostaining with ZO-1 antibody and by
transepithelial electrical resistance (TEER). Mice deleted for Sod2
in the RPE were gavage fed xaliproden daily for 4 months. SDOCT and ERG were used to analyze retinal structure. Optokinetic
measurements were used to assess visual acuity. Light and electron
microscopy were used to evaluate the retinal structure.
Results: Survival of cells following paraquat treatment was
dependent on the dose of xaliproden. An increase in the transcript
levels of NqO1, GSTM1, Nrf2, and metallothionein 1 was observed
with xaliproden treatment. The induction of IL-1b, IL-6 and VEGFA
in TNF-α treated cells was attenuated by xaliproden exposure.
TNF-α led to a decrease in TEER that was prevented by xaliproden.
Protection of the integrity of the tight junctions was also documented
by ZO-1 staining. Oral dosing with xaliproden induced the expression
of protective enzymes in the mouse retina. Mice with an Sod2
deletion in RPE treated with xaliproden showed improvement in
visual acuity and in thickness of the outer nuclear layer, but no
change in full field ERG amplitudes was observed among treatment
groups. These mice showed substantial vacuolization of the RPE and
disorganization of photoreceptor outer segments that was prevented
by oral treatment with the drug
Conclusions: Xaliproden can protect the RPE and retina against
oxidative as well as inflammatory insults, suggesting that it can be
developed as an oral drug for the treatment of AMD.
atrophic AMD, we are stating that this model can be used to test
therapies that support the retina in the absence of a supportive RPE.
Commercial Relationships: XiangDi Wang; Yunfeng Shi, None;
Monica M. Jablonski, UTRF (P)
Support: An unrestricted grant from Research to Prevent
Blindness, William and Ella Owens Medical Research Foundation,
University of Tennessee Research Foundation Maturation Grant,
Launch Your City Grant
Damage to the RPE and photoreceptors (left) is prevented by daily
p.o.dosing with xaliproden (right).
Commercial Relationships: Alfred S. Lewin, Shire, Inc. (C),
AGTC,Inc (C); Chulbul M. Ahmed, None; Manas Ranjan Biswal,
None; Hong Li, None; Pingyang Han, None
Support: BrightFocus Foundation
Intravitreal NA3 is neuroprotective in the RCS Rat
XiangDi Wang, Yunfeng Shi, Monica M. Jablonski. Ophthalmology,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: Age-related macular degeneration (AMD) is a debilitating
eye disease affecting as many as 25% of individuals past the age of
70 Western cultures. It initially presents as a degenerative disease
that does not directly affect photoreceptor cells, but rather it attacks
the supportive retinal pigment epithelial (RPE) cells. The purpose
of this investigation was to determine if asialo-triantennary (aka
NA3) provides neuroprotective support to the retina of the very
well characterized Royal College of Surgeons (RCS) rat. In RCS,
a mutation in Mertk renders the RPE unable to phagocytize outer
segment membranes, thus resulting in layer of outer segment debris
that fills the subretinal space and effectively removes the supportive
influence of the RPE upon the retina.
Methods: P21 RCS rats were dosed intravitreally in both eyes with
5µl of 2.5µM NA3 in PBS either once or twice per week for two
weeks. Control groups included PBS injections followed the same
time course, and no injections. Electroretinography (ERG), optical
coherence tomography (OCT) and visual acuity (VA) data were
collected at three time points: baseline before the start of treatment;
one week after first injection; and two weeks after first injection. Rats
were sacrificed after two weeks of therapy and eyes were enucleated.
One eye from each rat was embedded in plastic and thick sections
were cut to allow for histological analyses including measurement of
retinal layer thickness. The fellow eye is being prepared for GFAP
immunochemistry and TUNEL labeling.
Results: The structure and function of the retina was positively
affected by NA3. ERG amplitudes (a- and b-waves) and visual acuity
measurements were better preserved in NA3-treated eyes compared
to PBS-injected or no-injection control rats (p<0.05). The outer
nuclear layer thickness was also better preserved in NA3-treated
rats. Another physical change we saw was a better organization of
photoreceptor outer segments in eyes that were dosed with NA3.
Weekly NA3 injections were as or more efficacious than a twice
weekly dosing schedule.
Conclusions: Intravitreal NA3 treatment protected photoreceptor
structure and function in the RCS rat. It also preserved visual acuity.
These data suggest that NA3 may be an effective therapy for atrophic
AMD. While we are not claiming that the RCS rat fully mimics
Evaluating Reading Performance with a Head Mounted Aid for
Central Visual Field Loss
Anshul Gupta1, 2, Stephen Engel3, Erik J. Van Kuijk1,
Aurelie Calabrese3, Jacob Sanders3, Arthur Erdman2,
Gordon E. Legge3. 1Ophthalmology, University of Minnesota,
Roseville, MN; 2Mechanical Engineering, University of Minnesota,
Minneapolis, MN; 3Psychology, University of Minnesota,
Minneapolis, MN.
Purpose: We developed and tested a head-mounted, eye-tracker
based image enhancement aid for central field loss (CFL). The device
remaps the visual information lost to a simulated central scotoma in
real time, and displays it on a head mounted display’s (HMD) screen.
We used it to test the hypothesis that remapping lost information onto
a functional retinal location improves reading speed.
Methods: The device was comprised of a Sensics zSight HMD,
with a 60° diagonal field of view, fitted with a monocular Arrington
Research 60Hz eye tracker. The eye tracker provided gaze location
and enabled implementation of simulated central scotomas. The
images of text were remapped using a Gaussian Bump algorithm.
Remapped images were displayed on the HMD screen in real
time. Artificial scotomas subtending 4° and 8° were simulated on
7 normally sighted subjects, and reading speeds with and without
remapping were measured. For each condition, subjects silently read
7 MNREAD sentences with x-height of 0.83°. The line spacing was
set to 1.245° (1.5x). Reading speed was normalized by baseline speed
without a scotoma. Eye tracker calibration was assessed in real time,
and if calibration was lost, the trial was excluded.
Results: For the 8° scotoma with no remapping, subjects completed
13 of the 42, or 31% of the valid sentence trials, with an average
reading speed of 23% (± 1.6%) of their speed without a scotoma.
With remapping, subjects completed 46 of the 49, or 94% of the
sentences presented, with an average reading speed of 39% (±11.6%)
of their speed without a scotoma. The benefits of remapping are
clearly indicated by the large increase in completion rate and reading
speed. For the 4° scotoma, all subjects completed all trials. The
average normalized reading speed was 67% (±7%) of baseline with
remapping and 67% (±13%) without remapping. Remapping had no
significant effect on reading speed for this scotoma size.
Conclusions: Testing with normally sighted subjects with simulated
CFL suggests that the remapping device shows promise in improving
reading speeds in CFL patients, especially those with larger scotomas.
More testing is underway, and a future goal is to test on CFL patients.
lines cultured in the presence of pNAND can differentiate into mature
retinal epithelium and will be confirmed with immunocytochemistry.
Conclusions: Thermogelling pNAND copolymers have the potential
to function as an injectable cell delivery scaffold to improve the
survival and proliferation of transplanted cells in the retina. Further
investigations are necessary to evaluate efficacy in vivo.
Artificial Scotoma with and without Remapping
Commercial Relationships: Anshul Gupta, None; Stephen Engel,
None; Erik J. Van Kuijk, None; Aurelie Calabrese, None;
Jacob Sanders, None; Arthur Erdman, None; Gordon E. Legge
Therapeutic Cell Delivery Scaffold for the Retina
Megan Dodd1, Graeme Prosperi-Porta2, Heather Sheardown1, 2.
1
Chemical Engineering, McMaster University, Hamilton, ON,
Canada; 2Biomedical Engineering, McMaster University, Hamilton,
ON, Canada.
Purpose: Available treatments for retinal tissue degeneration
resulting in vision loss are unreliable, invasive, and do not address the
root cause of disease. Cell transplantation has been demonstrated as
a feasible approach for a lifelong treatment to repair and halt further
retinal degeneration, however, results have been mostly ineffective
and exhibit poor survival and integration. Thermogelling copolymers
can be optimized as cell delivery vehicles to enhance cell viability
and engraftment following sub-retinal administration.
Methods: N-isopropylacrylamide, acrylamide, acrylic acid
N-hydroxysuccinimide, and (r)-α-acryloyloxy-β,β-dimethyl-γbutyrolactone (pNAND) copolymers were synthesized using an
established method in different ratios (A,B,C). Bovine fibronectin
(FN) and/or phosphate buffered saline (PBS) were used to modify
the polymers and reduce the prevalence of reactive groups. The
temperature at which the polymer transitions to a gel (lower critical
solution temperature) was assessed using differential scanning
colourimetry [Fig. 1]. The viability and proliferation rates of human
and rat retinal pigment epithelial cells (ARPE-19) cultured in
2-dimensional (2D) and 3-dimensional (3D) copolymer gel constructs
were assessed with an MTT assay, and fluorescent cell imaging
[Fig. 2]. Differentiation of retinal pigment epithelial (RPE) cell lines
using established methods is currently underway, to be followed
with immunocytochemistry methods for detection of mature RPE
expression markers ZO-1, CRALBP, and RPE65.
Results: Synthesized pNAND copolymers and FN-pNAND
copolymers gel at physiological temperatures [Fig. 1]. Viability and
proliferation assays of ARPE-19 cells demonstrated that pNAND and
pNAND-FN prepared in 20% w/v PBS and DMEM provided viable
environments for 2D cell survival and proliferation, and 3D cell
survival in vitro [Fig. 2]. Preliminary results indicate that RPE cell
Commercial Relationships: Megan Dodd, None;
Graeme Prosperi-Porta, None; Heather Sheardown, None
Optimized Non-Viral Transfection of human RPE and IPE cells
used for a Gene-Therapeutic Treatment of neovascular AMD
Nina Harmening1, Gregg Sealy1, Martina Kropp1, Corinne Marie2,
Daniel Scherman2, Mattia Roncetti3, Pablo Aranda4,
Verónica Fernández4, Sandra Johnen5, Zsuzsanna Izsvák6,
Gabriele Thumann1. 1Ophthalmology, University of Geneva,
Geneva, Switzerland; 2INSERM U1022 - CNRS UMR8258, Unité de
Technologies Chimique et Biologiques pour la Santé, Paris, France;
3
IGEA SPA, Carpi, Italy; 43P Biopharmaceuticals SL, Noain Navarra,
Spain; 5Ophthalmology, University Hospital RWTH Aachen, Aachen,
Germany; 6Max Delbrück Center for Molecular Medicine, Berlin,
Germany.
Purpose: Basic research translation to the clinic mostly requires
development of reagents and equipment that meet requirements
of regulatory agencies. In two planned and EU-funded phase I/IIa
clinical trials to treat neovascular age-related macular degeneration,
autologous retinal (RPE) and iris pigment epithelial (IPE) cells
isolated from a patient, transfected with human gene of the pigment
epithelium-derived factor (hPEDF) will be transplanted subretinally
within a single surgical session. Transfection will be performed
using the Sleeping Beauty (SB100X) transposon system delivered in
pFAR4-miniplasmids, which are free of antibiotic resistance markers.
Since only limited number of cells are obtained from an iris or retina
biopsy, we have developed a protocol for high transfection efficiency
comprising novel buffers and modified electroporation device and
cuvettes. Here we report transfection results for primary human IPE
and RPE cells.
Methods: Primary human RPE and IPE cells were isolated from
donor eyes and transfected with hPEDF or Venus encoding pFAR4transposon plasmids. PEDF secretion was illustrated by Western Blot
analysis and quantified by ELISA. Fluorescence of Venus-transfected
cells was measured by image-based cytometry.
Results: Transfection efficiencies were significantly higher with
21.9%±8.2% for hRPE (n=13) and 26.9%±11.9% for hIPE (n=24)
using the novel buffer compared to 14.9%±6.4% for hRPE (n=13),
and 19.5%±12.2% for hIPE (n=24) using a commercial buffer.
With the newly developed cuvette, transfection efficiencies up to
24.7%±9.94% have been reached in hRPE cells (n=7). In cells
transfected immediately after isolation, the transfection efficiency
was 28.0%±34.1% for hRPE (n=19) and 20.5%±27.3% for hIPE
(n=12). PEDF secretion in hPEDF-transfected cells increased 2.9-fold
for hRPE cells (0.4±0.46 ng PEDF/h/104 cells) and 3.7-fold for hIPE
cells (0.4±0.26 ng PEDF/h/104 cells) compared to non-transfected
control cells.
Conclusions: The adapted transfection protocol comprising a
novel electroporation buffer, modified cuvettes, the use of pFAR4
miniplasmids and the SB100X transposon system made it possible to
deliver genes to primary cells reproducibly. The data reported here
show that PEDF-transfected cells express long-term elevated levels
of PEDF, meet the criteria for use of the transfection protocol for
human clinical trials.
Commercial Relationships: Nina Harmening, None;
Gregg Sealy, None; Martina Kropp, None; Corinne Marie, None;
Daniel Scherman, None; Mattia Roncetti, None; Pablo Aranda,
None; Verónica Fernández, None; Sandra Johnen, None;
Zsuzsanna Izsvák, None; Gabriele Thumann, None
Support: EU 7th Framework Program 305134
MicroRNA-155 inhibits polarization of macrophages to M2-type
and suppresses choroidal neovascularization
Xiaodong Sun, Pengfei Zhang. Ophthalmology/neurology, Shanghai
First Peoples Hospital, Shanghai, China.
Purpose: M2-type macrophages play great role in the development
of choroidal neovascularization (CNV). Thus, inhibition of M2
macrophage polarization may provide an alternative to anti-VEGF
therapy. This study evaluated the role of C/EBPβ in M2 macrophage
polarization. We also assessed whether microRNA-155 could inhibit
polarization of macrophages to M2-type via targeting C/EBPβ and
suppress CNV formation in laser-induced mice CNV model.
Methods: M2 macrophage polarization and the expression of C/
EBPβ were examined by Western blotting, real time PCR and
immunohistochemistry on day 1,3,7 after laser-induced CNV
formation in C57BL/6J mice. The effect of microRNA-155 on
M2 macrophage polarization was evaluated in the CNV model
by Western blotting, real time PCR and immunohistochemistry at
the same time points after intravitreal injection of microRNA-155
mimics. To evaluate the role of microRNA-155 on CNV
development, eyes of mice CNV model were examined by fluorescein
angiography, choroidal flatmounts on day 7 after intravitreal
injection.
Results: Arg-1+Ym-1+M2 macrophage and the expression of C/
EBPβ were significantly up-regulated in mice CNV model, while
microRNA-155 could inhibit Arg-1+Ym-1+M2 macrophage
polarization via targeting C/EBPβ in mice CNV model. Intravitreal
injection of microRNA-155 mimics inhibited CNV leakage and
neovascularization.
Conclusions: MicroRNA-155 modulating C/EBPβ plays great role
in M2 macrophage polarizaiton, while microRNA-155 mimics could
suppress CNV by inhibiting M2 macrophages polarization.
Commercial Relationships: Xiaodong Sun, None; Pengfei Zhang
Support: National Science Fund for Distinguished Young Scholars
81425006
A Phase 2 Study (EMERGE) Evaluating Repeated Intravitreal
Administration of ICON-1 in Patients With Choroidal
Neovascularization (CNV) Secondary to Age-related Macular
Degeneration (AMD)
Nancy J. Christmas. Colorado Retina Associates, Denver, CO.
Purpose: Current anti-VEGF therapies for wet AMD reduce
neovascular leakage and exudation but do not appear to reverse
the abnormal CNV progression in wet AMD. This Phase 2 study
examines the hypothesis that ICON-1, an anti-Tissue Factor (TF)
immunoconjugate protein, binds to pathologic vessels overexpressing
TF and acts via a new mechanism of action that can eliminate
abnormal CNV as well as inhibit the exudation either alone or in
combination with ranibizumab as compared to ranibizumab alone.
Methods: EMERGE is a randomized, double masked, active control
study in the United States. A total of 90 patients with treatment
naïve (in the study eye) CNV secondary to AMD are being enrolled.
Patient inclusion criteria include: ≥ 50 years of age, active primary
CNV due to AMD, Best Corrected Visual Acuity (BCVA) of 70 to 24
letters (worse than 20/40 and up to 20/320 Snellen equivalent) in the
study eye. Lesion characteristics include: a total lesion size <6 disc
areas (DA), CNV >50% of lesion size, presence of retinal fluid on
Optical Coherence Tomography (sdOCT). Patients are randomized
in a 1:1:1 ratio to receive intravitreal injections of ICON-1 (0.3 mg)
as monotherapy (n=30) or in combination with ranibizumab 0.5
mg (n=30) or ranibizumab 0.5 mg monotherapy (n=30). Patients
will receive 3 initial monthly injections followed by maximum 3
additional monthly injections based on protocol re-treatment criteria,
for a total of 6 months of treatment. The primary outcomes are mean
change from baseline in BCVA letter score and in Central Retinal
Thickness (CRT) at 3 months.
Results: The rationale for the study design, the mechanism of action
of ICON-1 and targeting TF, and detailed demographic characteristics
of enrolled patients will be presented.
Conclusions: The EMERGE study will provide important insight
into whether ICON-1 with its new anti-TF mechanism of action,
administered as monotherapy or in combination with ranibizumab,
leads to a modification of the CNV lesion along with reduction of
leakage, exudation and BCVA improvements, when compared to
ranibizumab alone.
Commercial Relationships: Nancy J. Christmas, Iconic
Therapeutics, Inc (R), Iconic Therapeutics, Inc (F)
Inhibition of choroidal neovascularization by siRNA targeting
tenascin-C
Yoshiyuki Kobayashi1, Shigeo Yoshida1, Yuki Kubo1, Yedi Zhou1,
Takahito Nakama1, Keijiro Ishikawa1, Shintaro Nakao1,
Yuji Oshima1, Akira Matsuda2, Tatsuro Ishibashi1, Koh-hei Sonoda1.
1
Ophthalmology, Kyushu University Graduate School of Medical
Sciences, Fukuoka, Japan; 2Ophthalmology, Juntendo University,
Bunkyo-ku, Japan.
Purpose: Tenascin-C has been reported to be highly expressed in
choroidal neovascular membranes (CNVMs) from patients with
age-related macular degeneration (AMD). However, its exact roles
of tenascin-C in the pathogenesis of CNVMs remain elusive. The
purpose of this study was to investigate the role of tenascin-C in
CNVM formation and to confirm the suppressive effect of tenascin-C
siRNA on mouse CNV model.
Methods: CNVMs of AMD patients and mouse CNV models were
examined immunohistochemically for the expression of tenascin-C,
α-SMA, pan-cytokeratin, CD31, and CD34. Tenascin-C mRNA
levels in human RPE cells treated with or without TGF-β2 (0, 1,
3, and 10ng/mL) were examined by real-time PCR. Proliferation
of human microvascular endothelial cells (HMVECs) treated with
tenascin-C was measured using bromodeoxyu-ridine (BrdU)-ELISA.
CNV volumes of tenascin-C knock-out mice and wild type mice
treated with vitreous injections of tenascin-C siRNA were measured
using isolectin-B4 staining.
Results: Double immunofluorescence analyses showed that
tenascin-C was co-stained with fibroblast-like RPE cells and vascular
endothelial cells in human CNVMs and mouse CNV. TGF-β2
induced tenascin-C mRNA expression in RPE cells. Tenascin-C
siRNA almost completely suppressed the TGFβ2-induced tenascin-C
expression. Tenascin-C promoted the proliferation of HMVECs. In
mouse CNV model, the mean CNV volume was significantly smaller
in tenascin-C knock-out group and tenascin-C siRNA injection group
compared with the control group.
Conclusions: Tenascin-C produced by RPE cells may play a role in
promoting CNV. siRNA targeting tenascin-C may have therapeutic
potential for inhibiting CNV.
Commercial Relationships: Yoshiyuki Kobayashi, None;
Shigeo Yoshida, None; Yuki Kubo, None; Yedi Zhou,
None; Takahito Nakama, None; Keijiro Ishikawa, None;
Shintaro Nakao, None; Yuji Oshima, None; Akira Matsuda, None;
Tatsuro Ishibashi, None; Koh-hei Sonoda, None
Support: Grant-in-Aid for JSPS Fellows from Japan Society for the
Promotion of Science (No.15J03433)
CRISPR/dCas9 to treat neovascular age-related macular
degeneration
Katie L. Pennington1, Sandeep Kumar2, Alex Jones3,
Margaret M. DeAngelis1, Yingbin Fu2, 1. 1Ophthalmology and Visual
Sciences, University of Utah Moran Eye Center, Salt Lake City, UT;
2
Department of Ophthalmology, Baylor College of Medicine Cullen
Eye Institute, Houston, TX; 3Department of Pathology, University of
Utah Huntsman Cancer Institute, Salt Lake City, UT.
Purpose: To develop a CRISPR/dCas9 system to act as an in vivo
therapeutic agent for treatment of AMD in a murine LASER-induced
choroidal neovascularization model.
Methods: Short guide RNAs (sgRNAs) were designed to target
mVEGF for transcriptional repression. The sgRNAs were tested for
repression efficiency using a dual luciferase assay. C57BL/6 mice
were subretinally injected with lentivirus encoding dCas9KRAB
and/ or sgRNA targeting mVEGF. Injected mice were subjected to
photocoagulation to induce choroidal neovascularization (CNV).
Treated eyes were harvested for analysis of either lesion size or gene
expression.
Results: Transfection of dCas9KRAB and sgRNAs targeting
mVEGF efficiently repressed expression of firefly luciferase under
the control of the mVEGF promoter + 5’ untranslated region (UTR)
in cell culture. Initial results indicate that subretinal injection with
dCas9KRAB and mVEGF sgRNA prior to LASER treatment reduces
neovascular lesion area.
Conclusions: Targeting mVEGF for transcriptional repression in vivo
using the dCas9KRAB-sgRNA system has potential as a therapeutic
method for the treatment of AMD.
Commercial Relationships: Katie L. Pennington, None;
Sandeep Kumar; Alex Jones, None; Margaret M. DeAngelis,
None; Yingbin Fu, None
Support: This work was supported by the National Institutes of
Health National Eye Institute (EY014800), and an Unrestricted
Grant from Research to Prevent Blindness, Inc., New York, NY, to
the Department of Ophthalmology & Visual Sciences, University of
Utah, with additional support awarded to Dr. DeAngelis from The
Skaggs Foundation for Research and the Carl Marshall Reeves &
Mildred Almen Reeves Foundation, Inc. NIH grant 1R01EY022901
awarded to Dr. Fu also supports this work.
Interspecific divergence of essential triad ribbon synapse protein,
pikachurin – implications for preclinical trials in photoreceptor
transplantation therapy
Christopher Laver, Joanne A. Matsubara. Ophthalmology and Visual
Sciences, University of British Columbia, Vancouver, BC, Canada.
Purpose: As photoreceptor transplantation rapidly moves closer to
the clinic, the importance of verifying graft efficacy in animal models
may have unforeseen xenogeneic barriers. Although conspecific
photoreceptor transplants have clearly resulted in functional
synaptogenesis, such unambiguous findings (ruling out false-positives
due to viral graft labeling or fusion) have not yet been shown for
discordant xenografts. From this, a fundamental question should be
raised: is xenosynaptogenesis likely between human photoreceptors
and mouse retina? The triad ribbon synapse between photoreceptor,
bipolar, and horizontal cells is unique and contains a trans-synaptic
protein essential and specific to its formation and function –
pikachurin. This protein is only produced by photoreceptors
and binds to an unknown target on the bipolar cell post-synaptic
terminus via its N-terminal domains. Thus, could interspecific
structural divergence be present that may inhibit this essential
trans-synaptic handshake?
Methods: In an effort to address this question computationally,
we compared pikachurin across placental mammals (1-to-1
orthologs of 35 species) using phylogenetic analysis, including dN/
dS measurements for positive selection (via PhyML and HyPhy
methods), and at the tertiary structural level using predictive
modeling (via Phyre2 and DeepAlign methods).
Results: Sequence similarity for human and mouse pikachurin were
77% and 94% in the N- and C-terminal domains, respectively. This
produced large structural divergence (41% length of alignment) in
the predicted models and was mainly localized to the N-terminal
domains. Selection analysis revealed strong positive (diversifying)
selection acting on the N-terminal domains (37 of the 61 positively
selected codons, PSCs, were found in this region). Negative
(purifying) selection was seen in the C-terminal domains, with the
exception of 20/24 C-terminal PSCs residing in a single LG-domain.
Together, these data indicate adaptive structural divergence in the
N-terminus among placental mammals.
Conclusions: Given the critical role of pikachurin in photoreceptor
graft-to-host synaptogenesis and function, and its structural
dissimilarity predicted between humans and mice, particularly in the
xeno-interaction domains, it is important to consider these factors
when designing future preclinical transplantation studies.
Commercial Relationships: Christopher Laver, None;
Joanne A. Matsubara
Polyphosphate protects against laser-induced choroidal
neovascularization in mice by blocking complement activation
and oxidative damage
Linnette M. Ocariza1, 2, Alice M. O’Byrne1, Jenny Huang1,
Stephanie A. Smith3, James H. Morrissey3, Jing Z. Cui4,
Joanne A. Matsubara4, Edward M. Conway1, 2. 1Centre for Blood
Research, University of British Columbia, Vancouver, BC, Canada;
2
Pathology and Laboratory Medicine, University of British Columbia,
Vancouver, BC, Canada; 3Biochemistry, University of Illinois,
Urbana, IL; 4Ophthalmology and Visual Sciences, University of
British Columbia, Vancouver, BC, Canada.
Purpose: The mechanisms underlying age-related macular
degeneration (AMD) are incompletely understood. However,
excess complement activation and oxidative stress are implicated.
We recently reported that polyphosphate (polyP), a naturally
occurring inorganic linear polymer, inhibits the terminal pathway of
complement. The goal of this project is to evaluate the efficacy of
administering polyP as a therapeutic agent in a laser-induced model
of choroidal neovascularization (CNV) in mice and to explore the
mechanisms of action of polyP in vitro.
Methods: Eyes of C57Bl6J mice were exposed to laser injuries that
induce CNV. PolyP130 or monoP (200 μM) was injected intravitreally
immediately after laser injury. Fourteen days later, eyes were
excised and dissected into eye cups. These were immunostained
for endothelial cells and deposition of the terminal complement
complex C5b-9, followed by flat mounting. Staining was quantified
by confocal microscopy and computer analysis. Cultured retinal
pigment epithelial cells (RPE) and choroid endothelial cells (CEC)
were exposed to H2O2 or to human serum to induce complement
activation. The effect of polyP and monoP on cell viability, catalase
expression, and complement activation was assessed by nuclear
staining, qRT-PCR, and deposition of C5b-9 detected by flow
cytometry, respectively. Statistical analyses were performed with One
Way ANOVA and Student’s t-test with significance at P < 0.05.
Results: Intravitreal injection of polyP130 exhibited a significant
reduction in C5b-9 deposition on the choroid following laser injury,
with a reduction in CNV, as compared to monoP. PolyP and monoP
protected the RPE and CEC from H2O2-induced loss of nuclear
integrity and increased catalase expression in a concentrationdependent manner. PolyP also suppressed C5b-9 deposition on the
cell surface.
Conclusions: In a murine model of wet AMD, polyP provided
protection by reducing CNV and complement activation. This is
in line with biochemical studies in which polyP interferes with the
terminal pathway of complement. We further showed that polyP
protects key cells associated with AMD from oxidative stress.
Further in vivo validation and toxicity studies are ongoing, and the
mechanisms by which polyP interferes with oxidative stress-induced
injury are being explored.
Commercial Relationships: Linnette M. Ocariza;
Alice M. O'Byrne, None; Jenny Huang, None; Stephanie A. Smith,
University of British Columbia (P), University of Illinois (P);
James H. Morrissey, University of British Columbia (P), University
of Illinois (P); Jing Z. Cui, University of British Columbia (P);
Joanne A. Matsubara, University of British Columbia (P);
Edward M. Conway, University of British Columbia (P)
Support: Canadian Institute for Health Research
Photobiomodulation induces drusen regression with
improvements in visual acuity and contrast sensitivity in subjects
with dry AMD
Graham Merry1, Robert Dotson1, Marion R. Munk2, Michael Walker4,
Robert Devenyi3. 1Ophthalmology, Photospectra Health Sciences
Inc, Toronto, ON, Canada; 2Dept. of Ophthalmology, Inselspital
University Hospital, Berne, Switzerland; 3Dept. of Ophthalmology,
University of Toronto, Toronto, ON, Canada; 4Walker Bioscience,
Carlsbad, CA.
Purpose: The aim of this study was to assess functional and
anatomical benefits of Photobiomodulation (PBM) using measures of
best corrected visual acuity (BCVA) and contrast sensitivity (CS) plus
changes in retinal drusen volume.
Methods: 42 eyes of 24 subjects with dry AMD, AREDS categories
2, 3, and 4 who had consented and been treated with PBM were
evaluated for ETDRS BCVA, CS and changes in drusen volume on
SD-OCT with linear mixed effects analyses.
Treatment parameters: LED light comprising of Red (670nm), Yellow
(590nm) and Infra-red (790nm) had been applied to the subjects eyes
over a 3 week course in an optimised regime.
Results: A statistically significant improvement of mean ETDRS
BCVA: +5.9 letters (P<0.001) and mean CS: +0.11 log units at 3
cycles per degree (P=0.02) was seen immediately following the
treatment and maintained for 3 months.
Significant mean Drusen Volume reduction of 0.024 mm3
(P<0.001) and Central Drusen Thickness reduction of mean 3.78
µm, (P < 0.001) was seen immediately following treatment and
maintained for 3 months.
Please see figures 1 and 2.
Conclusions: This is the first instance of statistically significant
improvement in both functional (BCVA and CS) and objective
anatomical (OCT drusen volume and central drusen thickness
reduction) findings reported in dry AMD subjects with PBM therapy.
The results indicate a sustained disease modifying effect following a
short non invasive treatment course.
This shows the percentage of eyes achieving up to one, two and three
ETDRS lines immediately post treatment.
This shows a representative example of an eye categorised as AREDS
3 with mainly convex, homogenous and low reflective drusen larger
than 125 µm. Baseline (Top) shows a drusen volume of 0.39 mm3
with a mean central 1 mm drusen thickness of 29 µm. Black numbers
indicate the mean drusen thickness of each ETDRS subgrid and red
numbers indicate the corresponding drusen volume (mm3). Bottom:
Follow up examination at 1 month. Overall drusen volume as well
as mean central drusen thickness has significantly decreased without
new formation of GA or disruption of the photoreceptor layers.
Commercial Relationships: Graham Merry, Lumithera Inc. (F),
Photospectra health sciences (I); Robert Dotson, Photospectra health
sciences (P); Marion R. Munk; Michael Walker, Lumithera Inc.
(C); Robert Devenyi, Lumithera Inc. (C)
A novel oral small molecule Factor D inhibitor blocks
complement activation in the blood and ocular tissues
Karen Anderson1, Sha-Mei Liao1, Juergen Maibaum2,
Frederic Cumin3, Omar Delgado1, Andrea De Erkenez1, Fang Liu1,
Upendra Argikar4, Ron Newton5, Bruce D. Jaffee1. 1Ophthalmology,
Novartis, Cambridge, MA; 2GDC, Novartis, Basel, Switzerland;
3
CPC, Novartis, Basel, Switzerland; 4MAP, Novartis, Cambridge,
MA; 5PCS, Novartis, Cambridge, MA.
Purpose: Strong genetic associations in humans have implicated
complement alternative pathway (AP) activation in age-related
macular degeneration (AMD). Factor D (FD) inhibition is proposed
as an attractive mechanism to reduce alternative pathway overactivity
at the first and rate-limiting step of the AP.
Methods: First-in-class, reversible, non-covalent Factor D inhibitors
were identified using structure-based drug design, employing a
tailored chemical library and in silico docking. Potency was assessed
initially in a biochemical enzymatic assay, followed by serumand whole blood-based in vitro functional assays using zymosan
to activate the AP and measuring deposition of membrane attack
complex (MAC). Compound 6 was investigated in vivo using a
humanized FD knock-in mouse pharmacodynamic (PD) model. In
this model, intraperitoneal lipopolysaccharide (LPS) was used to
activate complement, and AP breakdown products were monitored
in the blood and the eye tissue. We also evaluated the inhibitor’s
ability to block ex vivo serum complement activation after oral
administration of the compound to cynomolgus monkeys.
Results: The FD inhibitor 6 was very potent in 50% human whole
blood with a half-maximal inhibitory value (IC50) of 71 nM. The
compound dose-dependently inhibited LPS-induced generation of
Ba and iC3b/C3d in blood and eye after oral gavage in mice, with
complete suppression of the AP in both compartments at 100 mg/kg
PO. Dose dependent inhibition of ex vivo complement activation in
serum was also demonstrated after oral administration to monkeys,
with an IC50 value of 3.6 µM.
Conclusions: Compound 6 represents a first-in-class reversible,
selective, and potent small molecule FD inhibitor that is orally
efficacious in a humanized mouse model and in a monkey ex vivo
PD assay. Systemic dysregulation of the AP underlies a number of
human diseases with apparent local manifestations, like age-related
macular degeneration. Fully effective treatment of these diseases will
likely require inhibition at the systemic level. Our findings support
continued efforts aimed at developing oral AP-targeting therapies for
complement-mediated diseases.
Commercial Relationships: Karen Anderson, NIBR; ShaMei Liao, Novartis; Juergen Maibaum, Novartis; Frederic Cumin,
Novartis; Omar Delgado, Novartis; Andrea De Erkenez, Novartis;
Fang Liu, Novartis; Upendra Argikar, Novartis; Ron Newton,
Novartis; Bruce D. Jaffee, Novartis
Topical dorzolamide-timolol with intravitreal anti-vascular
endothelial growth factor for neovascular age-related macular
degeneration
Jayanth Sridhar, Jason Hsu, Abtin Shahlaee, Sunir Garg,
Marc Spirn, Mitchell Fineman, James Vander. Wills Eye Hospital,
Philadelphia, PA.
Purpose: To evaluate the effect of topical dorzolamide-timolol
on anatomic and functional outcomes in eyes with neovascular
age-related macular degeneration (AMD) with persistent exudation
despite fixed-interval intravitreal anti-vascular endothelial growth
factor (VEGF) injections.
Methods: A prospective single-arm interventional study was
performed. Patients with neovascular AMD and persistent macular
edema despite fixed-interval intravitreal anti-VEGF therapy were
enrolled. Baseline spectral-domain optical coherence tomography
(SD-OCT) and clinical data including visual acuity (VA) and
intraocular pressure (IOP) were obtained at enrollment and from
one visit prior to enrollment. Enrolled eyes were placed on topical
dorzolamide-timolol twice daily and continued to receive the same
intravitreal anti-VEGF therapy at the same interval as received prior
to enrollment for the duration of the study. Patients were followed
for two visits after enrollment. Central macular thickness (CMT),
maximum subretinal fluid (SRF) height, and maximum pigment
epithelial detachment (PED) height from SD-OCT were recorded
at each visit. Change in CMT was the primary outcome measure.
Change in maximum SRF height, maximum PED height, and VA
were the secondary outcome measures.
Results: Ten eyes of 10 patients completed the study. 8 eyes received
intravitreal aflibercept and 2 eyes received intravitreal ranibizumab.
All study eyes had been on chronic anti-VEGF therapy with the
same medication prior to study enrollment for an average of 21.9
injections. Mean CMT significantly decreased from 419.7 μm at
enrollment to 346.7 μm at the final visit (p=0.029). Mean maximum
SRF height decreased from 126.6 μm at enrollment to 62 μm at the
final visit (p=0.033) with complete resolution of SRF in 2 of 10 eyes.
Mean maximum PED height decreased from 277.4 μm at enrollment
to 227.8 μm at the final visit (p=0.13). Mean logarithm of the
minimum angle of resolution VA was 0.54 at enrollment and 0.49 at
final visit (p=0.62). Mean IOP in the study eyes decreased from 14.5
mm Hg at enrollment to 12.3 mm Hg at final visit (p=0.057).
Conclusions: Topical dorzolamide-timolol significantly reduced
macular edema and subretinal fluid in eyes with persistent exudation
despite consistent fixed-interval intravitreal anti-VEGF treatment for
neovascular AMD.
Commercial Relationships: Jayanth Sridhar, None; Jason Hsu,
None; Abtin Shahlaee, None; Sunir Garg, None; Marc Spirn,
None; Mitchell Fineman, None; James Vander, None
Support: Arch McNamara Research Fund
Clinical Trial: NCT02571972.
Response of RPE-Choroid Explants to Thermal Stimulation
Therapy of the Retinal Pigment Epithelium (TS-R)
Elisabeth Richert1, Stefan Koinzer1, Alexa K. Klettner1,
Ralf Brinkmann3, Jost Hillenkamp1, 2, Johann Roider1. 1University
of Kiel, Department of Ophthalmology, Kiel, Germany; 2University
of Würzburg, Department of Ophthalmology, Würzburg, Germany;
3
Medical Laser Center Lübeck, Lübeck, Germany.
Purpose: The TS-R is a sublethal laser technique for thermal
stimulation of the retinal pigment epithelium (RPE)-Bruchs
membrane-complex which avoids cell destruction of RPE and
neuroretina. The aim of this study was to investigate the influence of
TS-R on the release of AMD-relevant cell mediators such as MatrixMetalloproteinases (MMPs), Vascular Endothelial Growth Factor
(VEGF) and Pigment Epithelium Derived Factor (PEDF) using
different laser spot sizes.
Methods: Porcine RPE-Bruchs membrane-choroid explants were
irradiated with a 532 nm continuous wave laser and the threshold
of cell death was investigated by calcein staining. Explants were
irradiated with grids of sub-lethal spots (15 mW at 100 µm and 45
mW at 300 µm spot size, pulse duration 100 ms) and cultivated
in modified Ussing chambers which enabled the differentiation of
basal and apical cytokine release. The release of MMP 2 and 9 was
investigated by zymography and secretion of VEGF and PEDF was
analyzed by ELISA. Statistics were performed with Students T-test.
Results: Laser powers of 15 mW (100 µm spot size) and 45 mW
(300 µm spot size) applied over 100 ms did not induce cell death
in RPE cells. After TS-R with 300 µm spot size we observed a
significant increase of active MMP2 after 4 days to 1.56 fold of
control (SD±0.45, p<0.05) in basal compartments. The content of
PEDF increased significantly to 1.2 fold (SD±0.23, p<0.01) in treated
explants in comparison to controls while basolateral VEGF secretion
was not influenced by TS-R with 300 µm spots. Cytokine analysis
following TS-R with small 100 µm spots showed a significant
increase of PEDF to 1.28 fold (SD±0.32, p<0.01) compared to
control but no significant effect on MMP 2 and 9.
Conclusions: The TS-R represents a possible RPE stimulating
treatment for early AMD which could improve the flux across
Bruchs membrane. TS-R increases the basolateral release of active
MMP2 dependent on laser spot size, which might reverse age related
thickening of BrM. Basolateral VEGF secretion was not triggered by
TS-R while anti-angiogenic PEDF was increased by TS-R, indicating
an induction of an anti-angiogenic environment by TS-R.
Commercial Relationships: Elisabeth Richert, None;
Stefan Koinzer, None; Alexa K. Klettner, None; Ralf Brinkmann,
None; Jost Hillenkamp, None; Johann Roider, None
Support: Support of Bundesministerium für Bildung und Forschung
(BMBF) 13GW0043D
Subretinal fluid in Macular telangiectasia type 2 without
apparent choroidal neovascularization
Hemal Mehta2, 1, Simona Degli Esposti3, Catherine A. Egan3,
Mark C. Gillies2. 1Department of Ophthalmology, Royal Free
Hospital NHS Trust, London, United Kingdom; 2Macular Research
Group, Save Sight Institute, University of Sydney, Sydney, NSW,
Australia; 3Moorfields Eye Hospital, London, United Kingdom.
Purpose: To report a series of eyes in which subretinal fluid (SRF)
developed in macular telangiectasia type II (MacTel) in the apparent
absence of choroidal neovascularization (CNV).
Methods: Color fundus photography, optical coherence tomography
(OCT), and fluorescein angiography of patients in two of the largest
MacTel Study registries in the world at the Save Sight Institute in
Sydney and Moorfields Eye Hospital in London were assessed to
identify patients who have SRF causing a foveal detachment without
any other evidence of CNV. We confirm that the research followed
the tenets of the Declaration of Helsinki; informed consent was
obtained from the subjects; and the research was approved by local
institutional review boards.
Results: There were four female patients identified from the
registries with SRF in the apparent absence of CNV. Their ages
ranged from 50-66 years. Follow-up ranged from 5-8 years. Visual
acuity was only mildly affected by the presence of SRF. Occasionally,
the SRF resolved spontaneously, remaining stable for years before
recurring. There was variable response to intravitreal vascular
endothelial growth factor inhibitors (anti-VEGF) therapy.
Conclusions: These cases suggest that SRF in MacTel can
occur in the absence of CNV. We propose that deep intraretinal
neovascularization penetrates the subretinal space, leading to
accumulation of SRF. Intravitreal anti-VEGF therapy may not always
be necessary in such cases.
Commercial Relationships: Hemal Mehta; Simona Degli Esposti,
None; Catherine A. Egan, None; Mark C. Gillies, None
Long-term clinical outcomes after intravitreal bevacizumab
injections or photodynamic therapy for myopic choroidal
neovascularization: 7 years follow-up
Eui Chun Kang, Minkyo Kim, Kyou Ho Lee, Hyoung Jun Koh.
Department of Ophthalmology, Institute of Vision Research, Seoul,
Korea (the Republic of).
Purpose: To compare the long-term clinical outcomes after
intravitreal bevacizumab (IVB) injections or photodynamic therapy
(PDT) for myopic choroidal neovascularization (CNV) through 7
years follow-up.
Methods: In this retrospective comparative case series, a total of
37 eyes with 37 patients which were treated with IVB (17 eyes) or
PDT (20 eyes) were included. Preoperative classification for myopic
maculopathy was made according to the international photographic
classification. The primary outcome was the mean change of
best-corrected visual acuity (BCVA) between two groups from the
baseline through 7 years follow-up.
Results: The mean change of BCVA from baseline to 7 years followup was greater in the IVB group compared with the PDT group
(-0.177±0.316 vs. 0.091±0.417 logMAR, P = 0.044). The BCVA
from baseline through 7 years follow-up improved in the IVB group
(0.642 ± 0.427 vs. 0.463 ± 0.439 logMAR, P = 0.029), but did not
improve in the PDT group (0.645 ± 0.402 vs. 0.736 ± 0.576 logMAR,
P = 0.266). In subgroup-analysis with 18 eyes with preoperative
tessellated fundus only (category 1), there was no difference in
improvement of BCVA (-0.338 ± 0.277 vs. -0.156 ± 0.184 logMAR,
P = 0.166) and progression of CRA (0.17 ± 0.47 vs. 0 mm2, P =
0.264), CSFT (250.3±14.6 vs. 247.8±38.8 µm, P = 0.929) and SFCT
(140.6±74.8 vs. 146.3±104.9 µm, P = 0.929) between two groups at
7 years follow-up. However, 17 eyes with preoperative diffuse CRA
(category 2) showed no change of BCVA in IVB group, but decreased
BCVA in PDT group (-0.033 ± 0.289 vs. 0.338 ± 0.105 logMAR, P =
0.03). Although, the CSFT was not different between groups (237.7 ±
61.6 vs. 197.8 ± 55.4 µm, P = 0.171), the progression of CRA (1.88 ±
2.89 vs. 6.43 ± 4.44 mm2, P = 0.019) and thinned SCFT (44.3 ± 38.2
vs. 16.9 ± 16.3 µm, P = 0.028) was less in IVB group compared with
PDT group.
Conclusions: The mean change of BCVA was superior in
eyes treated with IVB compared to PDT for 7 years follow-up.
Additionally, the decreased BCVA and the progression of CRA
after PDT treatment were more prominent in eyes with preoperative
diffuse CRA (category 2) compared with IVB injections.
Commercial Relationships: Eui Chun Kang, None; Minkyo Kim,
None; Kyou Ho Lee, None; Hyoung Jun Koh, None
Outcomes of Hydrocortisone Treatment on the Distribution of
Junctional Proteins and Subretinal H20 Transport Regulators in
RPE Cells
Martin Rudolf, Mahdy Ranjbar, Armin Mohi, Salvatore Grisanti,
Ayseguel Tura. Ophthalmology, University of Luebeck, Lübeck,
Germany.
Purpose: Glucocorticoids are known to have a negative effect on
subretinal fluid accumulations in central serous chorioretinopathy
(CSC). This disease involves typically the RPE/choriocapillaris
complex but the exact pathophysiology is still unidentified. Here we
wanted to evaluate the effects of Hydrocortisone on the integrity of
cellular junctions and the major regulators of subretinal H20 transport
in RPE cells.
Methods: Adult human RPE cells (Passages 1-3) were seeded
on to poly-L-lysine-coated 8-well chamber slides and allowed to
differentiate in low serum (2% v/v) medium for at least 8 weeks.
Porcine RPE-Choroid-Sclera explants were preincubated overnight
on culture inserts (0.45 µm pore size) with the RPE-side facing
upwards. Cells and the basal side of explants were incubated with
Hydrocortisone (0-5-50-500 nM) for 1 day. Protein distribution was
analyzed by immunostaining and -blotting.
Results: Hydrocortisone led to a significant increase in the levels
of beta-Catenin, N-Cadherin, and ZO-1 at cellular junctions and the
expression of Na/K-ATPase in human RPE cells. A considerable
increase was detected in the levels of Na/K-ATPase and the
water channel Aquaporin-1 in both the apical and the basolateral
membranes as well as the expression of Na/K/2Cl cotransporter
(NKCC1) in the lateral membrane of RPE cells in porcine explants
incubated with Hydrocortisone.
Conclusions: Exposure to Hydrocortisone can strengthen the
integrity of RPE junctions while disrupting the polarized, mainly
apical distribution of Na/K-ATPase. The latter event might lead to
a failure in the establishment of the ionic gradient essential for the
transepithelial H2O transport towards choroid. This may in turn result
in an increased accumulation of subretinal fluid in CSC and by that
in a worsening of the disease, especially if this mechanism is already
significantly compromised by CSC pathophysiology.
Commercial Relationships: Martin Rudolf, None;
Mahdy Ranjbar, None; Armin Mohi, None; Salvatore Grisanti,
None; Ayseguel Tura, None
Macular Pigment and Carotenoid Supplementation in Patients
with Pattern Dystrophy
Eileen Hwang, Christopher D. Conrady, Jim Bell, Paul S. Bernstein.
Ophthalmology, Moran Eye Center, Salt Lake City, UT.
Purpose: The role of nutritional supplementation in pattern
dystrophy is not known, yet many patients with pattern dystrophy are
taking carotenoid supplements. We performed a case-control study
comparing patients with pattern dystrophy to controls to determine
whether pattern dystrophy was associated with a deficiency in
macular pigment. We also studied macular pigment levels in pattern
dystrophy patients with and without supplementation to assess
whether oral supplementation resulted in higher levels of carotenoids.
Methods: Nine patients were identified with pattern dystrophy.
Diagnostic criteria included a fundus appearance consistent with
adult vitelliform dystrophy or butterfly dystrophy and a subretinal
hyperreflective lesion on optical coherence tomography. Patients
with drusen were excluded. Nine age-matched control patients were
selected from the retina clinic population. The Heidelberg Spectralis
with macular pigment optical density software was utilized to
measure macular pigment by dual wavelength autofluorescence.
Results: The mean macular pigment volume under the curve
(MPVUC) within 2 degrees of the fovea in patients with pattern
dystrophy was 2700 +/- 1300 (optical density * pixels +/- standard
error). In the control population, the value was 2900 +/- 1400. There
was no significant difference between the two groups (p = 0.66).
MPVUC measurements ranged widely from 1172 to 4945 in the
pattern dystrophy population, but this did not appear to depend on
supplement intake. The mean MPVUC for the four pattern dystrophy
patients not on carotenoid supplements was 2239 +/- 1247, and the
mean MPVUC for the five pattern dystrophy patients on supplements
was 2999 +/- 1339 (p=0.41). None of the control patients were taking
supplements.
Conclusions: Pattern dystrophy did not appear to be associated with
low MPVUC as measured by two-wavelength autofluorescence.
Patients with pattern dystrophy taking carotenoid supplements may
not have higher MPVUC than pattern dystrophy patients not taking
supplements. This suggests that carotenoid supplementation may
not be useful in pattern dystrophy, but larger studies are needed to
determine whether supplementation is clinically beneficial in this
population.
Commercial Relationships: Eileen Hwang, None;
Christopher D. Conrady, None; Jim Bell; Paul S. Bernstein, None
Support: Achievement Rewards for College Scientists (ARCS), NIH
National Eye Institute R01 EY011600 ,Core Grant 14800, Research
to Prevent Blindness
Retinal function and structure are preserved in a canine model
of CLN2 disease after intravitreal implantation of stem cells
genetically modified to overproduce TPP1 enzyme
Rebecca E. Whiting1, Christopher J. Tracy2, Jacqueline W. Pearce3,
Lauren E. Gillespie1, Baye G. Williamson3, Daniella P. Vansteenkiste3,
Joan R. Coates3, Jeffrey N. Bryan3, Martin L. Katz1. 1Ophthalmology,
University of Missouri School of Medicine, Columbia, MO;
2
Genetics Area Program, University of Missouri, Columbia, MO;
3
Veterinary Medicine & Surgery, University of Missouri, Columbia,
MO.
Purpose: CLN2 disease is an inherited, childhood disorder which
causes progressive vision loss, and cognitive and neurological
dysfunction. Dogs with CLN2 disease exhibit similar clinical signs
including progressive retinal degeneration, beginning at 4 to 5 months
of age, characterized by significantly reduced electroretinogram
(ERG) b-wave amplitudes and multi-focal retinal detachments. CLN2
disease results from lack of the lysosomal enzyme, TPP1. Studies
were performed to determine whether supplying the deficient TPP1
enzyme with intravitreal ex vivo gene therapy can prevent retinal
degeneration in CLN2-affected dogs.
Methods: Autologously derived mesenchymal stem cells (MSCs)
from CLN2-affected Dachshunds (n=5) were transduced with adenoassociated virus (AAV2)-packaged DNA constructs that direct stable
overexpression and secretion of TPP1 or stable expression of green
fluorescent protein (GFP). TPP1-transduced cells were implanted
into the vitreous of one eye and GFP-transduced cells into the
contralateral eye at 3 months of age. Thereafter, dogs were evaluated
monthly with bilateral ERG and in vivo retinal imaging until near
end-stage neurologic disease was reached around 10 months of age.
Results: For each dog, the eye treated with TPP1-expressing MSCs
had better preserved retinal function and structure compared to the
control eye, which had deficits typical of untreated CLN2 disease.
Treatment delayed the onset of ERG amplitude decline by 1 to 2
months and reduced the rate of decline thereafter by an average of
41% (range: 23% to 67%) compared to that of the control eye. The
control retina developed extensive detachment lesions in 4 of 5 dogs,
while lesions were fully prevented in 2 of the contralateral TPP1treated eyes and greatly reduced in the remaining treated eyes.
Conclusions: We have demonstrated the potential to inhibit retinal
degeneration with a continuous supply of therapeutic compound
produced by autologous, genetically modified stem cells that have
been implanted into the vitreous. This approach to therapeutic drug
delivery offers a long-term treatment and is applicable to many other
hereditary retinal degenerative diseases.
TPP1-expressing MSCs preserve retinal structure. In a 7-month-old
dog, 23% of the control retina exhibits detachment lesions, while
lesions are fully prevented in the TPP1-treated retina.
Commercial Relationships: Rebecca E. Whiting, None;
Christopher J. Tracy, None; Jacqueline W. Pearce, None;
Lauren E. Gillespie, None; Baye G. Williamson, None;
Daniella P. Vansteenkiste, None; Joan R. Coates, None;
Jeffrey N. Bryan, None; Martin L. Katz, None
Support: NIH Grant 1R01EY023968; Knights Templar Eye
Foundation

Session 414 AMD Post-VEGF Novel Therapies

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