El principio del fin de la ceguera.

Archivos diarios: 11/06/2013

If your interests lie in macular and vitreoretinal disease, then this year’s ARVO meeting surely did not disappoint. For the first time, a full-day retina subspecialty symposium was held just before ARVO—giving retinal specialists and general eye care practitioners an additional opportunity to hear about the latest and greatest in retina from a roster of world-renowned experts.

It was the perfect way to gear up for the many sessions at ARVO that reviewed the constantly evolving landscape of treatments and imaging technology and introduced new avenues of retinal research. Attendees were eager to hear the long-awaited results of the Age-Related Eye Disease Study 2 (See “The Latest on AREDS2 at ARVO 2013.”). Other hot topics this year included the role of Eylea in age-related macular degeneration (AMD), the genetics of AMD and several novel treatment approaches to AMD. Presentations also highlighted a new retinal prosthesis that may offer help to patients with retinitis pigmentosa (RP), as well as new ways to treat macula edema.

Retinal Prosthesis

It was a milestone moment for retinal technology in February when the FDA approved the first implanted device to treat adults with advanced RP. The Argus II Electronic Retinal Prosthesis System (Second Sight Medical Products)—which has been cleared in Europe since 2011—should be commercially available in the US later this year. For now, it’s been granted “humanitarian use,” an approval pathway limited to devices that treat or diagnose fewer than 4,000 people in the US each year.

The Argus II Implant (left) attaches to the retinal surface with a tack. The cable that both powers the chip and conducts the image signal from the episcleral housing is seen temporally. An early frame of a fluorescein angiogram (right) in a patient with the Argus II Implant demonstrates some persistent macular perfusion. Images: Elaine Leibenbaum, Julia Haller, MD, and Carl Regillo, MD.

A few studies evaluated this prosthesis, hopefully paving the way for its more widespread use. One study looked at the safety profile of 16 patients in Europe who had received the implant.1040/C0017 The patients were followed on average for 6.2 months, and reported no surgical or serious, device-related adverse effects. Ten patients experienced no surgery or device-related adverse events at all, whereas the other six reported minor adverse effects, such as IOP elevation, nausea, fainting, conjunctival irritation and a retinal tear.

A second study found that the Argus II implant has good long-term reliability, with only one failure in 30 subjects (each with an average of 4.2 years of use, representing more than 125 cumulative patient years).1037/C0014 Further, accelerated lifetime testing demonstrated that finished implants have more than a 10-year lifetime in accelerated testing. Another study confirmed these results, echoing previous tests that demonstrated the ability of the prosthesis to provide visual function over several years.349

Diabetic Retinopathy

Researchers evaluated 759 patients from the RISE/RIDE Phase III trials to see if Lucentis (intravitreal ranibizumab, Genentech) had an effect on the severity of a patient’s diabetic retinopathy.4028 Results showed that a greater proportion of patients in the ranibizumab arm had a two- or three-step regression of diabetic retinopathy on the ETDRS scale vs. those in the sham group. A three-step improvement was achieved at 36 months in 3.3% of the sham group, compared to 15.0% and 13.2% in the 0.3mg and 0.5mg treated eyes, respectively. Over the course of 36 months, 33.9% of the sham-treated eyes developed proliferative diabetic retinopathy, as opposed to only 12.8% and 15.1% of the ranibizumab-treated eyes.

Another study evaluated the safety and efficacy of Macugen (intravitreal pegaptanib, OSI Pharmaceuticals Inc.) combined with panretinal photocoagulation (PRP) vs. PRP alone in the regression of retinal neovascularization in eyes with high-risk proliferative disease.2439/C0140 At six months, the combination of pegaptanib with PRP showed better preservation of best-corrected vision, greater decrease in retinal thickness and maintained visual field better than PRP alone, but showed no major difference in neovascular regression.

A second study of 30 patients compared combination therapy of ranibizumab with PRP vs. PRP alone in treatment-naïve proliferative diabetic retinopathy (PDR).5761/D0008 This study uncovered a greater change in best-corrected vision, a larger decrease in central retinal thickness and a lower incidence of vitreous hemorrhage in the combination treated group––again suggesting that anti-VEGF agents in conjunction with PRP may be preferred to PRP alone.

Additionally, a retrospective study of 78 patients seemed to indicate that metformin may reduce the rate of PDR in type 2 diabetes patients.2249/C0150 In the non-metformin group, 15 patients (45.5%) developed PDR as compared to just 12 patients (27.3%) in the metformin-treated group, indicating a trend of less PDR in the metformin-treated group. A larger study is recommended.

Macular Edema

Several studies are investigating alternative approaches to treat macular edema––either secondary to diabetes or vein occlusion. The MOZART study evaluated the safety and efficacy of an intravitreal dexamethasone implant (Ozurdex, Allergan) in 59 patients with visual impairment from diabetic macula edema (DME).2387/C0088 Investigators noted that, over the six months, central retinal thickness was reduced and acuity improved—28% of patients had 20/40 or better acuity vs. just 6% at baseline. They observed IOP greater than 25mm in 7% of patients, with 4% of patients developing cataracts. No endophthalmitis was reported.

A second study showed positive results using a different intravitreal dexamethasone implant injection (DEX-I), with a gain of more than 10 letters in 27% of cases at two months and 24% at four months.2382/C0084 However, this study showed that recurrence of edema was observed in 76% of cases at four months, leading to re-treatment in more than one-third of cases.

Diabetic macular edema, as confirmed by optical coherence tomography.

Another study evaluated Ozurdex in patients with macular edema from vein occlusions. Forty eyes were treated with Ozurdex and were followed for six to 24 months.254/D0099 Overall, 94% showed initial regression on OCT, lasting an average of 4.2 months, with two lines of improvement. Overall, 59% improved 14.2 letters on average, while 10% worsened and 31% remained the same. Approximately 19% had elevated IOP and were treated with drops. In eyes that were not previously treated, the results were even better—86% showed improvement. Half of the patients required retreatment, with an average of 1.6 treatments per year. The results seem to indicate that Ozurdex may be an effective treatment in such patients––even those who did not respond well to anti-VEGF agents.

Other research looked at the role of laser, as well as anti-VEGF in combination with laser, in the treatment of macular edema. The LLOMD study evaluated 15 eyes of 13 patients with reduced visual acuity secondary to diabetic macular edema who had a mean VA of 20/100.2396/C0097 At six months, the mean VA gain was 12.6 ETDRS letters, with the central retinal thickness decreasing an average of 76.7µm in patients who received laser in combination with ranibizumab. Additionally, 37.5% of patients required a second injection at six months. However, with the addition of laser, the study showed that the number of injections needed over the first year was greatly reduced compared to previous studies of injections alone. In total, the researchers determined that approximately 10 injections are needed during the first year. Further, they concluded that adding macula grid laser to ranibizumab injection may reduce the economic burden of treatment.

Two additional studies revealed that reduced-energy focal macular photocoagulation could have advantages over traditional focal macular laser. 2375/C0076,2416/C0117 Both seemed to indicate that, by reducing the laser exposure when performing the procedure, there were decreases in CRT and increases in vision––with potentially less collateral damage and inflammation to surrounding viable tissue. More research is needed to investigate whether reduced-energy focal macular photocoagulation could replace more traditional laser therapy as the standard.

Eye on Eylea

Several reports evaluated Eylea (aflibercept, Regeneron Pharmaceuticals), the latest FDA-approved anti-VEGF agent for the treatment of wet AMD. A number of these looked at the role of Eylea in patients whose choroidal neovascularization did not respond to other agents, namely Lucentis (ranibizumab, Genentech) and Avastin (bevacizumab, Genentech/Roche).

One study evaluated 41 eyes of 34 such patients—77% of these patients had a good response to Eylea after one month, demonstrating decrease in central retinal thickness and absorption of subretinal fluid.4176/A0094 Best-corrected visual acuity improved in these patients to 20/74, from 20/122.5 at baseline.

A second study evaluated 60 eyes of 52 patients that did not respond after five consecutive injections of the other agents.3806/B0116 After three Eylea injections, 28 eyes (46.7%) displayed improved acuity, while 18 eyes (30%) showed decreased acuity, and 14 (23.3%) had no change in acuity at three months.

Lastly, a study evaluated 19 eyes of 17 patients receiving Eylea as primary therapy, with dosing as needed.3817/B0127 Over a 20-week period, patients received on average 1.84 injections, with an interval between injections of approximately 11 weeks. Five of these patients were determined to be non-responders to other anti-VEGF agents. Of these five, four responded positively to Eylea, indicating again that Eylea may be an effective alternative for patients who do not respond to other agents. Also, this study seems to indicate that the interval to repeat injections may be longer with Eylea than the other agents.

However, a separate study looked at the costs associated with Eylea.3838/B0148 The researchers hypothesized that, despite fewer injections, the cost of treatment per patient would actually increase. The study reviewed the records of 30 patients treated for wet AMD from 2011 to 2012 at the Cincinnati Eye Institute. The average duration between Avastin or Lucentis injections was 29 days, as opposed to 34 days with Eylea injections. No complications were noted in any groups. Total cost over the six months was $3,700 for Avastin, $96,000 for Lucentis and $366,300 for Eylea. This study suggests that while Eylea may reduce the frequency of injections, office visits and possibly complications, it appears to add considerable health care costs per patient.

New AMD Treatments

Several studies evaluated novel treatments for AMD. One study investigated the safety and feasibility of an episcleral brachytherapy device (SMD-1) for wet AMD.3787/B0097 Six patients received radiation for five and a half minutes to the macular CNV using a brachytherapy probe adjacent to the macular sclera via a subtenon retrobulbar approach. Patients also received concomitant anti-VEGF injections, as needed. The procedure was readily performed and well tolerated, with no adverse effects. At three months, all patients experienced improved best-corrected vision, with a mean gain of 19 ETDRS letters. At 12 months, three patients continued to demonstrate improved vision of seven letters on average, and two of those patients did not require any additional injections. All patients had reduced macular thickness compared to baseline, but two patients did demonstrate a reduction in vision.

Another study evaluated the safety of 1% CLT-005 topical eye drops, designed to inhibit Stat3, which has been associated with neovascular and inflammatory processes in animal studies.1716 The researchers determined that the drug was able to deliver the active ingredient to the RPE/choroid in animal eyes, without adverse effects—paving the way for additional studies regarding its role in the treatment of AMD or geographic atrophy (GA).

Australian researchers looked at the progression of early AMD after treatment with nanosecond pulse laser compared to patients with a natural history of AMD.4146/A0064 They treated 48 patients with bilateral high-risk AMD with ultra-low energy laser in 12 spots around the macula of one eye. At 12 months, three of the 48 treated participants progressed to GA, while seven of the 70 control group progressed. At 24 months, four in the treated group and nine in the control groups progressed to GA, suggesting that a single course of nanosecond laser intervention may potentially reduce the odds of progression to advanced AMD. A larger randomized controlled study is now underway.

Another study evaluated the safety and tolerability of an extrafoveal subretinal injection called rAAV.sFlt-1, an anti-VEGF gene therapy for AMD, in elderly patients.4504 Twelve patients underwent the procedure with minor adverse effects and no evidence of local or systemic toxicity. The researchers noted that this injection should be further evaluated as a potential strategy for long-term anti-VEGF therapy.

Research continues on Emixustat HCL, a novel orally administered agent in development for the treatment of GA associated with dry AMD.4506 Emixustat HCL is a rod visual cycle modulator that inhibits isomerase activity and reduces retinal toxins, such as A2E, which damages the RPE and overlying photoreceptors. Four dose levels and two dose regimens were examined in 72 patients who were followed for 90 days. No adverse systemic effects of concern were noted, with just two patients experiencing treatment-related events. All ocular adverse effects resolved upon drug cessation, and were mild with no severe events observed. Results were encouraging, and a long-term Phase II study in now underway to evaluate its role in GA patients.

Other studies looked at using existing therapy more effectively. A team of researchers in Italy evaluated whether ketorolac eye drops combined with ranibizumab intravitreal injections would provide additional efficacy over ranibizumab alone in wet AMD.4175/A0093 Sixty eyes were divided into two groups: one received ranibizumab alone, and one was treated with ranibizumab plus ketorolac BID for six months. At the end of six months, there was no statistically significant difference in best-corrected vision or number of injections required. However, the mean six-month change in central macular thickness was 146.53µm in the combination group, while the change was 106.88µm in the ranibizumab-only group. This is the first study to identify an additional effect of ketorolac eye drops combined with ranibizumab. More studies would be needed before a change in current protocol would be appropriate.

Two separate studies evaluated photodynamic therapy in combination with anti-VEGF injections.4509,3790/B0100Both indicated that this therapeutic combination might be an effective way of improving acuity in patients with wet AMD, while perhaps reducing the overall number of treatments needed. In one of the studies, 96.2% of eyes lost fewer than 15 letters, and 27.3% gained 15 or more letters.

Genetics in AMD

Genetics in eye care have been garnering a lot of attention lately, specifically the role of genetics in AMD.

One study evaluated data from the 100 Genomes Project to confirm the contribution of known genetic risk factors for AMD.6166/C0051 This investigation revealed that, in the population of European descent, CFH has the largest attributable risk (25.6%), followed by ARMS 2 (22.5%), then C3 (9.1%) and CST3 (5.8%). In other populations, the risk allele in ARMS2 is the major contributor to risk, followed by CFH. In Asian and African populations, CST3 takes precedent over C3 as the third strongest contributor to AMD risk.

This patient is at high risk for AMD due to multiple confluent drusen in both eyes. Perhaps genetic testing could one day identify patients like this earlier.

In Spanish patients, a study found that CFH and CB genes, combined with environmental risk factors such as smoking and body mass index, were associated with an increased risk of GA.6183/C0068 A second abstract confirmed the role of CFH gene in AMD risk in a cohort of Brazilian AMD patients.6175/C0060

In another study evaluating the genetic contribution of AMD in 38 Armenian patients, researchers found no genetic differences in the risk alleles compared to a Caucasian population.6196/C0081 All of this research indicates the genetic factors that could influence the development or AMD may be very similar across different groups.

Interestingly, some of these same studies seem to suggest that the HDL-related CETP gene may be associated with AMD in African Americans, pointing to a potential risk modifier in lipid pathways.6168/C0053

An abstract submitted by Johanna Seddon, MD, ScM, identified three new genes that may add to the predictive power of risk models for progression to advanced AMD.6178/C0063 They are the R1210c mutation in CFH, and variants to the genes COL8A1 and RAD51B. She suggested that these new genes will be useful for AMD surveillance in the future, along with genes that have already been identified and established factors such as drusen size, baseline AMD status, demographics and environmental factors (including smoking, age and body mass index).

Additional studies attempted to see if there was a link between genetic profile and response to treatment. One study evaluated the genetic profile of 835 patients from the CATT (Comparison of AMD Treatment Trial) trial to determine if certain genotypes responded better to treatment than others.6187/C0072 Results revealed there were no strong associations between the studied genotypes and response to anti-VEGF treatment.

A second study evaluated the IVAN study and also was unable to find any associations between genetic profiles and response to anti-VEGF treatment.6185/C0070 However, another study of 43 patients seemed to indicate that patients with high-risk alleles for AMD responded more poorly to treatments than those with low-risk alleles.6186/C0071

This link of genetic profiles to treatment response may continue to be investigated, as this could bring us closer to personalized treatment of AMD––based on genetic factors and other components.

Retinitis Pigmentosa and Rare Diseases

Usher syndrome (USH) is the most frequent cause of inherited deafness–blindness in humans, accounting for approximately 50% of all cases and affecting one child out of 25,000. Today, there is no specific and/or curative therapy for USH patients, except the hearing aids and cochlear implants designed to correct hearing impairment. Among the three USH clinical subtypes defined by the severity of the hearing impairment, the presence/absence of vestibular dysfunction, and the age of retinitis pigmentosa (RP) onset, USH1 is the most severe. In the past decade, several strategies to prevent and treat RP have been developed, including retinal implants, pharmacological agents, stem cells, retinal cell transplantation, and gene therapy. Clinical trials for some of these strategies are currently underway.

In 2012, the first ever gene therapy for USH1B, UshStat® (developed by Oxford BioMedica and using its LentiVector® platform technology) moved into human studies. Three dose levels for safety, tolerability and aspects of biological activity of UshStat® are under evaluation at the Oregon Health & Science University’s Casey Eye Institute where the study has started and at the Centre Hospitalier Nationale d’Ophthalmologie des Quinze-Vingts in Paris where the study should start soon with support from Foundation Fighting Blindness. This safety study will prepare for future efficacy trials. Further treatment advances will require more scientific effort such as: 1) to identify all causative genes and determine their function, 2) to develop appropriate animal models, 3) to uncover the mechanisms underlying the retinal defect in USH syndrome, 4) to engineer appropriate viruses for transfer of genetic material into appropriate cells.

We have started a new clinical trial using gene therapy with an adeno-associated viral (AAV) vector encoding Rab escort protein-1 (REP1) to treat patients suffering from choroideremia (NCT01461213). An AAV2 vector encoding human REP1 driven by a CBA promoter with a woodchuck hepatitis virus post-translational regulatory element (WPRE) was used. The first six patients have reached six months follow-up and a paper detailing the effects of gene therapy is currently undergoing peer review. The formal results of the study will therefore be reported soon. In the meantime we can share the following observations:

Through microperimetry testing, we have observed an underlying functional defect in this disease, similar to Leber congenital amaurosis (LCA), but more subtle. In fact, this observation was made previously by Dr. Sam Jacobson using psychophysical testing in choroideremia patients. This observation is exciting because it implies that we might see improvements in retinal sensitivity (and visual acuity in later stages) as evidence of successful gene transfer.

We have observed no problems in detaching the fovea in these six patients, or at least any negative effects on their vision are more than compensated for by gene expression relating to the vector. Retinal thinning was only seen in one of the six patients, but in a non-seeing area and stretching of this area was noted intra-operatively. We believe the problems of foveal thinning in the LCA studies relate to a combination of patients having a thin fovea at baseline and the injection being initiated too close to the fovea and/or too rapidly, thereby causing excessive horizontal stretch of the neurosensory retina. The technique we have developed would be suitable for all the other rod-cone dystrophies where the peripheral retina is thinner than the central macula.

We have set the study up as multicentre, so that expert ophthalmologists from other UK centres follow the patients up after six months. This spirit of openness is ideal as other experts have the opportunity to examine the patients and therefore provide independent verification of our initial findings. We have also been sharing our data with other centres worldwide to help them submit regulatory applications.

Leber congenital amaurosis (LCA) refers to a form of inherited retinopathy with early-onset and severe loss of vision. Clinical trial of gene augmentation therapy for LCA caused by RPE65 mutations has been ongoing at the University of Pennsylvania and University of Florida since 2007. Earlier reports from our group, as well as from other groups performing similar clinical trials in parallel, showed that a single surgical procedure introducing the normal version of the RPE65 gene leads to improved vision in a matter of days to weeks. But RPE65 –LCA is a complex blindness due to two pathologies: progressive loss of photoreceptors to degeneration, and malfunction of all surviving photoreceptors. The assumption all along was that correction of the malfunction would halt or slow down the photoreceptor degeneration.

To evaluate this natural assumption, we imaged the retina in patients and measured the sublayer within the retina where photoreceptor nuclei reside. This sublayer slowly thins over many years and the rate of thinning should mirror the rate of photoreceptor degeneration. In untreated eyes, the photoreceptor layer measurements were abnormally thin even in the youngest of the patients with ages as early as 3 years, and showed progressive further thinning when examined serially over 5 years. The rate of thinning was about 10% per year. We compared the rates of photoreceptor degeneration in treated regions to untreated regions and found no difference. The treated regions continued along the expected rate of degeneration, even though paradoxically retaining the vision improvement achieved immediately after the gene therapy.

We hypothesized that once initiated, retinal degeneration advances despite successful gene augmentation therapy. We tested this hypothesis in the dog model of the human disease. But first we needed to know the natural history of degeneration in the dogs. Examination of a large number of dog eyes with the same non-invasive imaging tools used in human patients showed that dog retinas are without any degeneration for the first 5 years of their lives (until ~35 human years). Thus RPE65-disease is late onset in the dog compared to the human. When dogs were treated at ages after the onset of degeneration, gene therapy resulted in improved visual function but did not slow down the retinal degeneration – just like the results in patients.

At this stage we do not know why the photoreceptor cells degenerate in RPE65-disease but our results are most consistent with the following speculative explanation for the paradox observed. Visual function is originating from the minority of cells that are functionally-potent wheras degeneration is dominated by the loss of cells that are functionally-silent. In order to improve outcomes of gene therapy, we need to start using stages of animal models that truly represent the human condition. We need to assume less and prove more. We need to better understand the pathways of cell loss and find means to augment gene augmentation by inducing cell-protective pathways or inhibiting cell-death mechanisms.

UF-021, isopropyl unoprostone, is an eyedrop which was already approved to treat eyes with glaucoma or ocular hypertension in the USA and Japan. Previous studies showed that topical IU increases human choroidal blood flow, and that an intravitreal injection of IU protects photoreceptors from light damage in rats. It was also shown that apoptosis of cultured photoreceptors was successfully inhibited by unoprostone but not by prostaglandin.

A Phase 2 clinical trial has been completed in Japan, in which 103 Japanese RP patients were randomized into 3 groups: high dose, low dose, and placebo groups. They completed 6 months of follow-up time. The primary endpoint was central retinal sensitivity measured by microperimetry. The secondary endpoints were: visual acuity, contrast sensitivity, retinal sensitivity by HFA, and vision-related QOL assessed by VFQ25. In a comparison of changes from baseline within groups, there was a statistically significant increase in central retinal sensitivity threshold for the high-dose group. In a post-hoc analysis that adjusted for baseline differences, a dose-dependent improvement in mean central sensitivity was demonstrated. There were statistically significant differences between the placebo and high-dose groups in the change from baseline in central retinal sensitivity and in the proportion of patients with worsening of the retinal sensitivity by ≥4dB (placebo 21.2%, high dose 2.6%). There was also a statistically significant change from baseline in the high-dose group in mean retinal sensitivity by HFA. A subgroup analysis showed that, among subjects whose central retinal sensitivity <29.4dB, there was a significant difference in the high-dose group. In a comparison within groups of changes from baseline in patients’ VFQ-25 total points values, the changes within the high dose group were statistically significant. In conclusion, this phase 2 trial shows that UF-021 improves or maintains the central retinal sensitivity in RP patients.

Recently, a Phase 3 clinical trial has started in Japan in which 300 RP patients will be randomized into 2 groups: high-dose and placebo groups. The primary endpoint of P3 is the central retinal sensitivity measured with HFA. All patients will be followed for 52 weeks. After this period, they will enter an open label safety study for 52 weeks.

One of the most common causes of irreversible blindness is loss of photoreceptor cells. An exciting new therapeutic strategy for treating these conditions, which include AMD and diabetic retinopathy as well as inherited retinopathies, is photoreceptor cell transplantation.

Some years ago we demonstrated that it is possible to transplant photoreceptor cells into an adult mouse retina, provided the cells are at a particular stage of development – a post-mitotic photoreceptor precursor (MacLaren et al., Nature in 2006). Even though we could only manage to obtain around 1000 integrated cells, this was an important proof-of-concept and forms the basis of our programme because this knowledge might be used to generate appropriate cells for transplantation from stem cells.

After 5 years of optimization, we were able to increase the efficiency of transplantation and with around 30- 40,0000 integrated cells we could demonstrate restoration of vision in a mouse model of stationary night blindness (Pearson et al, Nature 2012). This is another important proof-of-concept because it demonstrates that the transplanted photoreceptor cells make functional connections and there is enough plasticity to actually improve vision. Recently, we have also shown that we can transplant photoreceptor precursors in a variety of animal models of retinal degeneration including in models of severe degeneration and still improve vision (Barber et al, PNAS 2013).

So far we have focused primarily on studies involving the transplantation of photoreceptor precursors obtained from early post-natal retinas into visually-impaired adult mice. In order to develop this into a useful treatment we need to use a renewable source of cells for transplantation. Embryonic stem cells (ESC) represent the most promising such source of cells for transplantation and considerable progress has been made in their differentiation in the laboratory toward photoreceptor lineages. For some years now we have been trying to differentiate mouse ES into photoreceptor precursors efficiently enough to be able to transplant effectively. Until very recently we have not been successful. However, we have now used a new differentiation protocol based on the landmark paper in Nature in 2011 by Yoshiki Sasai. He demonstrated it is possible to generate synthetic retinae from mouse ESCs.

We have now optimised this protocol and shown for the first time that following transplantation, the rod precursors from these ESC-derived retinae, integrate and mature within adult degenerate retinae. This is an important study because it shows conclusively that ESCs can provide a useful source of photoreceptors for retinal cell transplantation.

Now that we have shown that it is possible to transplant mouse ESC- derived photoreceptors, the next step towards clinical translation is to develop human ESC lines to provide a potentially unlimited source of transplantation-competent photoreceptor precursors. We are now starting to work with hES cells and aim to develop GMP compliant processes that may enable translation to clinical trials.

6) The Use of Muller Glial Cells in Retina Repair. Dr. Tom Reh Dept. of Biological Structure, University of Washington, Seattle, WA, USA.

The study of Regenerative Medicine attempts to find ways to replace cells in the body that have degenerated such as do photoreceptor cells in retinal degenerative diseases like retinitis pigmentosa and age-related macular degeneration. The use of stem cells is one such method that takes an undifferentiated (usually embryonic) cell and converts it into a mature, functional cell such as a photoreceptor neuron that would be found in the adult retina.

Other methods though can be used to generate new photoreceptors, one of which utilizes Muller glial cells that are natural support elements in the retina. Over the past 10 years now, Dr. Reh and his collaborators have provided evidence that the retinas of some higher species such as the chicken had the potential to generate new neurons. In response to an acute insult that extensively damaged the photoreceptors, he found that many of the Muller cells re-entered the cell cycle and began to express biochemical markers associated with embryonic retinal progenitor cells. Work in vitro continues on this to dissect the various factors involved in the reprogramming of glial elements into neurons. Differentiation of the glial-derived progenitor cells offers several advantages over more traditional forms of transplantation and stem cell implantation. For example, the cells are already in place with no need to surgically implant new cells. Also, there is the possibility of decreasing or eliminating the deleterious immunological problems evoked by implantation of foreign cells into the retina.

In lower species such as fish and amphibians, there is a well known ability to regenerate retinal neurons after trauma or degeneration induced by other means. Understanding the molecular and biochemical pathways of regeneration could ultimately lead to the ability to reprogram native glial cells into retinal neurons such as photoreceptor cells in the human.

We have previously demonstrated in dogs with CNGB3-achromatopsia that intravitreal bolus injection of CNTF (1) resulted in transient restoration of cone function and day vision, and (2) optimized long-term cone functional response to AAV-mediated gene augmentation therapy. The objective of this study was to determine if sustained intravitreal delivery of CNTF by encapsulated cell technology (ECT) could reverse the disease phenotype of CNGB3-achromatopsia in dogs long-term.

Dogs homozygous for the D262N missense mutation in CNGB3 were unilaterally implanted with CNTF-secreting, encapsulated cell implants. The pre-implant CNTF secretion rate is 15 ng/day. The animals were 3 months (n=2) and 27 months (n=1) of age and were day blind with no recordable cone ERG prior to surgery. Following implant placement, the dogs were examined weekly by standard full-field electroretinography under general anesthesia and visual behavioral testing in an obstacle avoidance course.

In the operated eyes, day vision and cone function were partially restored by 1 week following CNTF-implant placement. The amplitudes of single and flicker cone ERG responses were small (~5-10% of normal) but were maintained for at least 5 weeks thus far. Scotopic ERG responses were reduced in 2 of the 3 implanted eyes to <30% of amplitudes recorded in the non-operated fellow eyes. These ERG data were comparable to our observations following single intravitreal bolus injection of 12 μg CNTF.

In conclusion, sustained intravitreal delivery of CNTF by ECT rescues cone function and day vision in CNGB3-achromatopsia. It remains to be shown if this therapeutic effect can be sustained long-term and if ECT can be combined with AAV-mediated cone-directed gene augmentation to optimize treatment.

To date, several clinical studies have been completed, including a phase 1 study in RP, 2 phase 2 studies in RP (early and late stages) and a phase 2 study in GA. Cone photoreceptor preservation by AOSLO was demonstrated in the RP study.

A phase 1 study in MacTel was completed recently and the study showed that both NT-501 implant and surgical procedure were well tolerated. We are actively planning a multi-center phase 2 study for MacTel in the U.S. and Australia.

The primary endpoint of the phased 2 study will be “change in area of IS/OS loss at 2 years post implant as measured by en face imaging by SDOCT in study eye(s)”.

The study will include 68 subjects

Study duration will be 2 years

We expect to initiate the study in the second half of 2013.

From a regulatory perspective, we have achieved the following objectives:

Obtained the fast track status for RP and GA with the FDA

Obtained the orphan designation for MacTel and RP with both the FDA and EMA.

Received agreement from FDA and EMA regarding the primary endpoint for the MacTel phase 2 study

Actively pursuing agreement/advice from the FDA and EMA regarding using cone preservation by AOSLO as the primary endpoint for the RP phase 3 studies

Note: The new results reported below are taken from a recent press release (May 16, 2013) from Advanced Cell Technology.

Human stem cells have two important characteristics. First, their numbers can be expanded in cell culture to almost unlimited amounts. Secondly, the cells have the potential of developing into any cell type of the body, for example, retinal photoreceptor or retinal pigment epithelial (RPE) cells. Thus, stem cell implantation is attractive as a possible therapy in replacing dead or defective cells in cases of retinal degeneration.

In macular diseases such as Stargardt Disease and Age-Related Macular Degeneration (AMD), early problems in pigment epithelial cell function can lead to death of photoreceptor cells. Thus, replacement of dead or defective RPE cells through stem cell implantation could prolong photoreceptor cell life and even restore their function.

The company Advanced Cell Technology (ACT) is conducting clinical trials in patients with Startardt Disease and AMD and have already published preliminary results indicating the safety and tolerability of their stem cell implantation. In this report (Lancet 379:713, 2012), the hESC-derived RPE cells showed “no signs of hyperproliferation, tumorgenicity, ectopic tissue formation or apparent rejection after 4 months.” In their most recent press release, ACT reposts that vision of one of their patients “has improved from 20/400 to 20/40 following treatment.” Although ACT adds the disclaimer that “improvement in the patient’s vision reported in this press release may not be indicative of future results….”, the positive results are welcome and give hope for improvement in other patients using this form of Regenerative Medicine.

The eye is an organ that is well-suited for the development and testing of novel therapeutic approaches. It is easily accessible and allows local application of therapeutic agents with reduced risk of systemic effects. Need exists for the development of non-viral therapeutic approaches for ocular diseases. Our lab and others have investigated the potentials of nanotechnology for ocular delivery of therapeutic genes. Investigations thus far have highlighted great potentials for nanoparticles to be a successful approach for ocular gene delivery.

Our group has focused on the efficacy of compacted DNA nanoparticles for the treatment of different diseases, particularly those associated with the retina and retinal pigment epithelium. We have shown that nanoparticle treatment leads to efficient transfection of ocular cells, long term gene expression, and exerts no toxic effects on the eye even after multiple injections. These nanoparticles mediate significant functional rescue in models of retinitis pigmentosa, Stargardt macular dystrophy and Leber’s congenital amaurosis. They have no limitations on the size of the genetic cargo and effective gene expression has been demonstrated with vectors up to 20 kb in the lung and 14 kb in the eye, making them an ideal complement to AAVs especially for delivery of large genes. Furthermore, in a side-by-side comparison study with AAV, we recently reported that nanoparticles can drive gene expression on a comparable scale and longevity to AAV.

We have synthesized two series of orally active multifunctional antioxidants (MFAOs) possessing distinct free radical scavenging activity and independent metal attenuating activity. Both series demonstrate similar selective metal chelating activity against iron, copper and zinc, as well as similar antioxidant activity against hydroxyl, peroxide and superoxide radicals assessed in human retinal pigmented epithelial (ARPE-19), human neuroblastoma (SH-SY5Y), and SRA human lens epithelial cells. Oral administration to mice indicates that the first MFAO series rapidly accumulates in the lens and retina but not the brain. Rat studies indicate that this MFAO series delays lens changes induced by diabetes, gamma irradiation, and UV irradiation and protects the photoreceptor layer against light damage. In contrast to the first series, oral administration of the second series of MFAOs to mice results in their accumulation in the brain and retina, but not the lens. Both series of MFAOs demonstrate no toxicity when administered by gavage at doses of 1600 mg/kg.

Since mitochondrial dysfunction and amyloid beta (Aβ) neurotoxicity are associated with age-related retinal changes, the effect of MFAOs on these factors has also been investigated in human neuroblastoma and retinal pigmented epithelial cells. Although these compounds chelate iron, they do not adversely affect mitochondrial function. In fact, they actually protect mitochondria from manganese poisoning. Both MFAO series also bind zinc; but zinquin staining indicates that these compounds do not adversely reduce cytoplasmic zinc levels. However, both MFAO series readily remove zinc from the neurotoxic amyloid beta zinc complex which is not readily degraded by matrix metalloproteinase (MMP)-2. The removal of zinc from the neurotoxic amyloid beta zinc complex by MFAOs allows MMP2 to degrade amyloid beta. The interaction of MFAOs with zinc is similar to the “metal attenuation” activity demonstrated by clioquinol and reported for PBT2, an analog of clioquinol undergoing a Phase 3 clinical trial for the treatment of Alzheimer’s dementia.

The Age-Related Eye Disease Study 2 (AREDS2), a multi-center randomized clinical trial tested the addition of lutein (10 mg)/zeaxanthin (2 mg) and/or omega-3 fatty acids to the original AREDS formulation. The investigators found neither harmful nor beneficial effects of omega-3 fatty acids for the treatment of age-related macular degeneration (AMD). The main effects analyses indicated that lutein/zeaxanthin had beneficial effects for reducing the risk of advanced AMD by 10%, for reducing the risk of advanced AMD by 26% in persons with the lowest dietary intake of lutein/zeaxanthin, and for reducing the risk for progression to neovascular AMD by 22% in the head-to-head comparisons of lutein/zeaxanthin vs. beta-carotene.

The safety of the AREDS formulation was tested with the elimination of beta-carotene. The finding of increased lung cancer in those supplemented with beta-carotene in mostly former smokers provided compelling data to eliminate beta-carotene. Furthermore, there was an increased efficacious effect in those treated with lutein/zeaxanthin compared with beta-carotene as indicated above. A safer and more efficacious formulation, which can be defined as the AREDS2 formulation would eliminate beta-carotene, add lutein (10 mg) and zeaxanthin (2 mg), and retain vitamin C (500 mg), vitamin E (400 international units), zinc (80 mg) and copper (2 mg).

a) Epiretinal implant: The ARGUSII system is produced by Second Sight Medical Products, Sylmar, California with sixty epiretinal electrodes, goggles with camera and an electronic transmission system to the back of the eye. After a study in thirty patients had been completed, the system had received the CE-mark and FDA approval recently. Twenty more patients had been implanted in the meantime. Maximum visual acuity reported so far was 20/1200 with mobility improving in most patients. Presently, a post-marketing study is planned and the costs for the device have been mentioned as 150.000 US$ per piece.

The camera has been reported to have some advantage because of the possibility of zooming but also has disadvantages as fading of the image occurs easier and can be compensated only with head nodding. For further information see Humayun et al. (2012) Ophthalmology 119:779–88.

b) Subretinal implant : The Alpha IMS is produced by Retina Implant AG, Tübingen/Reutlingen, Germany. After a pilot study in eleven patients (2005-2009) a clinical main trial with presently 25 more patients is ongoing in several centers (Oxford, London, Tübingen, Dresden, Hong Kong among other centers). This system consists of a light sensitive chip, similar to a camera chip that is implanted into the subretinal space in the back of the eye at the position of the degenerated photoreceptors. Each chip consists of 1.500 light sensitive photodiodes, amplifiers and electrodes. The image is resolved point by point and, depending on the brightness of each point, a current is forwarded to the bipolar cell layer. There is no camera outside the eye because all the electronics are in the eye and move with the eye except for the power supply coil that is implanted under the skin behind the ear. The best recorded visual acuity is 20/546. Due to microsaccades, fading occurs rarely and facial recognition has been reported by some patients. For further data see Stingl et al. (rspb.royalsocietypublishing.org/content/280/1757/20130077.full.pdf+html). The study is still ongoing and observation time so far is 1.5 years.

c) Other developments: In Australia, three patients have received a wire bound suprachoroidal (Bionic Vision) electrode array with 24 electrodes; no complete implant is available yet. Another new development includes preclinical work with passive elements by Palanker et al. Stanford University, USA (Mathieson et al. (2012) Nature Photonics 6, 391-7). By means of having three photosensitive elements per pixel in a row, sufficient voltage can be produced in order to stimulate the retinal neurons. However, this requires an enormous amount of light that only can be produced by special laser driven goggles.

A newly founded company in Paris (PIXIUM VISION) has now joined forces with former German-Swiss company IMI and the Palanker group to explore further possibilities of the use of passive elements.

For further comparison on the various approaches see Zrenner (2012), Nature Photonics 6: 344–5.

Degenerative blinding diseases such as retinitis pigmentosa and age-related macular degeneration affect millions of patients around the world. These disorders cause the progressive loss of rod and cone photoreceptors in the retina, eventually leading to complete blindness. A number of approaches are being explored to restore vision to blind patients. Our goal is develop and test novel pharmacological therapies for vision restoration. Here, we demonstrate the restoration of visual function to blind mice following the injection of red-shifted chemical “photoswitch” compounds.

We have created several small molecule photoswitches that can be used to control the activity of neurons by reversibly blocking native ion channels in response to light. In order to evaluate the ability of these photoswitches to restore light sensitivity to blind mice, we have tested them in an rd1 mouse model of retinitis pigmentosa. Our in vitro retinal light response measurements were carried out using a multi-electrode array (MEA). We also tested the restoration of several visually guided behaviors in blind mice in vivo.

We have previously demonstrated that the photoswitch AAQ could drive light responses in formerly blind retinas in vitro as well as restore the pupillary light reflex and light-aversive behaviors in blind mice. Here, we present the restoration of light sensitivity to blind mice in vitro and in vivo with two red-shifted photoswitch molecules, DENAQ and BENAQ. Unlike AAQ, DENAQ and BENAQ do not require the use of ultraviolet light and render a blind retina sensitive to visible (blue-green) light at a light intensity equivalent to ordinary daylight. These red-shifted molecules photosensitize the retinas of blind rd1 mice in vitro. The photoswitches persist up to several weeks in vivo and are well tolerated in the eye. DENAQ and BENAQ are selective for degenerated rather than healthy retinal tissue, suggesting they would not interfere with any remaining photoreceptor mediated vision in patients with retinal diseases. Intravitreal injection of DENAQ also restores light sensitivity to rd1 mice in vivo in an exploratory locomotory behavioral assay. Furthermore, DENAQ-injected rd1 animals are able to reverse the polarity of their naïve light response after appropriate fear conditioning, indicating their restored vision is sufficient for visual learning to take place.

In conclusion, red-shifted chemical photoswitches such as DENAQ and BENAQ, and our pharmacological approach in general, hold great promise for restoring visual function in end-stage degenerative blinding diseases.

Our project aims at restoring vision in patients with blindness consecutive to photoreceptor degeneration as in retinitis pigmentosa using optogenetic proteins. It is a collaborative program supported by Foundation Fighting Blindness and Gensight, a start-up company created thanks to the support of Novartis and Novartis Venture fund. The project is achieved together with Dr Roska at FMI in Basel Switzerland and Pr Sahel and myself at the Vision Institute in Paris France. Retinal prostheses have shown that it is possible to restore some vision in blind patients but we work on an alternative strategy, the optogenetic therapy, which consists in reactivating residual retinal cells by expressing photosensitive ionic pumps or channels after the photoreceptor degeneration thanks to gene therapy. We are trying to reactive photoreceptors, which have lost their sensitivity to light using the chloride pump, halorhodopsin.

Indeed, we have shown:

1) that some cone PRs remain in blind patients affected by retinitis pigmentosa. These PRs have lost their photosensitive part, the outer segment, which explains why the patient is blind. Similar dormant or non-photosensitive PRs are also found in animal models of the disease.

2) and that these dormant PR in blind mice can be reactivated to light by expressing halorhodopsin, via gene therapy. Visual perception in these blind mice is indicated by light responses in PR and retinal ganglion cells.

3) After showing these results on blind mice, we have used postmortem human retina in culture to show that human cone PRs can express the functional halorhodopsin at a sufficient level to polarize PRs

Our current objective is to demonstrate that this high level of expression can be obtained in vivo on non-human primates and that it does not trigger an immune response because halorhodopsin is a bacterial protein. We have already tested our AAV viral vector and obtained high and selective expression of the Green fluorescent protein GFP in cone PRs. When we co-express halorhodopsin and GFP, we still maintain a high protein expression as indicated by the GFP fluorescence.

We still need to demonstrate that this expression level can activate the retinal tissue by itself and measure markers of the immune response prior to testing the clinical batch of virus. This demonstration is quite complex to obtain because we are using normal monkeys. Therefore, their PRs have their natural response to light and we are introducing an additional sensitivity to light. We have therefore to bleach the natural light response to demonstrate an optogenetic-mediated response. To give us more chances, we can also culture the retina for some period of time to lose the natural response and demonstrate the halorhodopsin-elicited response.

Inherited retinal diseases (RD) display an impressive degree of allelic and genetic heterogeneity as nearly 10,000 mutations in >190 genes have been identified. Mutations in these genes account for 30% to 90% of cases, depending on the type of disease. Comprehensive genotyping of persons with inherited RD improves genetic counseling and the accuracy of disease prognoses. Moreover, genotyping identifies persons who are eligible for novel therapies. We are entering an era of routine testing for RD-associated defects, both in academic and non-academic centers. The identified known and novel variants are not published or deposited in open access databases. Sharing sequence variants and their associated phenotypes are at the heart of DNA diagnostics and it therefore is of utmost importance to register this information in publicly available databases.

The structure and use of RD-mutation databases need to meet the following criteria: 1). Web-based open access; 2). Registration of all published sequence variants; 3). Easy upload of new variants; 4). Accurate assessment of mutation data; 5). Regular updating. We propose the implementation of Leiden Open Variation Databases (LOVDs) for all RD genes in the next five years. LOVDs were previously created for 10 Usher syndrome-associated genes. A team of BS students and staff members in Islamabad, will collect all published sequence variants for the remaining RD genes, scrutinize them for their proper annotations, and upload them in gene-specific LOVDs. World-wide curators will check the new entries.

‘Empty’ LOVDs were created for all RD associated genes, and all published variants were registered for AIPL1, LCA5, RDH5, SEMA4A, and TULP1. Other mutation repositories previously were created for CEP290, NDP, and Bardet-Biedl syndrome-associated genes. These will be taken up in LOVDs. In 2013, variants of another 20 RD-associated genes will be deposited in LOVDs and the existing LOVDs will be updated every year.

The long-term success of this endeavor relies on a robust organization of sequence variant updating, proper curation, database maintenance, and a sound financial basis. It will also be vital to introduce compulsory deposition of sequence variants prior to publication submissions, and the compliance of diagnostic facilities worldwide to deposit unpublished variants in LOVDs.

C) RI Announcements, New Business and Conclusion

RD 2014 – First Announcement – Dr. Matthew LaVail

The XVIth International Symposium on Retinal Degeneration will be held in Pacific Grove, California, USA on July 13-18, 2014. The venue will be the Asilomar Conference Grounds. The organizers are: Drs. Catherine Bowes Rickman, Matthew LaVail, Joe G. Hollyfield, Robert E. Anderson, John Ash and Christian Grimm. The RD2014 website is under construction; please continue checking for when it is up and running. It is hoped that up to 30 Travel Awards can be funded for students, Post-doctoral Fellows and junior faculty members below the rank of Associate Professor. Important deadlines are: 1) Travel Award applications: February 3, 2014 2) Meeting and Hotel Registration: March 17, 2014 3) Online Abstract submission: March 17, 2014

4) Final Comments – Ms. C. Fasser

Ms. Fasser thanked all the speakers for their excellent presentations.

She remarked how heartening it is to see all the progress in clinical trials for the inherited retinal degenerative diseases and to hear about new trials to come in the future.

She hoped that all participants would have a very fruitful ARVO meeting and that we would all meet again for the RI meeting next year in Orlando.