The Foundation Fighting Blindness. Stargardt s Disease- Moving to Clinical Trials

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1 The Foundation Fighting Blindness Stargardt s Disease- Moving to Clinical Trials On Friday, April 6, 2007, a symposium was held at the Coleman Center in New York under the auspices of the Foundation Fighting Blindness. The symposium was co-chaired by Gerald A. Fishman, M.D., and Rando Allikmets, Ph.D. A list of participants and agenda is included at the end of the document. The intended purpose of the meeting was to address issues relevant to moving pre-clinical studies of Stargardt disease into clinical trials. Introduction Stargardt disease, initially described by the German ophthalmologist Karl Stargardt in 1909, is predominantly an autosomal recessively inherited disorder, although an autosomal dominant Stargardt-like phenotype has also been described. The estimated frequency of the recessive form is 1 out of 8,000 to 10,000 in the United States. The dominant form of dystrophy is approximately ten times less frequent than the recessive form. For the purpose of this document, the disease described in 1963 as fundus flavimaculatus, by the Swiss ophthalmologist, Adolphe Franceschetti, and recessive Stargardt disease are considered as synonymous. Mutations in the ABCA4 gene have been identified in the recessive form of Stargardt disease. The dominant form of Stargardt-like dystrophy is associated with mutation in a different gene, ELOV4. Mutations in the ABCA4 gene have also been found in 50-60% of patients with autosomal recessive cone-rod dystrophy and in a small fraction ( 5%) of patients with a panretinal dystrophy, in many cases described as a severe form of atypical retinitis pigmentosa. Patients with Stargardt disease develop a progressive impairment of visual acuity which can begin, most frequently, within the first or second decades of life. A segment of patients will develop symptoms of visual acuity loss as late as the fourth, or even fifth decade of life. The visual symptoms are accompanied by atrophic-appearing lesions within the macula, various degrees of yellowish-white lesions at the level of the retinal pigment epithelium (RPE), which are referred to as fundus flecks, or other changes indicative of degenerative changes of the photoreceptors and the RPE. The clinically apparent fundus changes are preceded by excess accumulation of lipofuscin in the RPE. This abnormal accumulation can be indirectly observed with autofluorescence imaging taking advantage of the natural autofluorescent property of lipofuscin. Patients will also, not infrequently, complain of a delay in their ability to function visually when they move from brightly to dimly lit environments, such as walking

2 2 into a movie theater. This subjective complaint can be objectively measured with psychophysical dark adaptation testing which quantifies the time course of improvement of vision following presentation of a bright light. In Stargardt disease, the extent of dark adaptation delay has been shown to relate to the severity of the retinal dysfunction. Disease Mechanism in ABCA4-Associated Stargardt Disease The ABCA4 gene encodes the ABCR protein (also called rim protein) which is located in the outer segments of rod and cone photoreceptors. In vitro studies, together with results from a knockout mouse model, support the hypothesis that ABCR serves as an outwardly directed flippase by transporting all-trans-retinal (as a Schiff base conjugate with phosphatidylethanolamine, N-retinylidene-PE, NRPE) from the intradiscal portion of an outer segment disc membrane to the cytoplasmic portion where it is hydrolyzed to all-trans-retinol. In the absence, or inefficient functioning, of the ABCR protein, a second molecule of all-trans-retinal reacts with phosphatidylethanolamine to form the phosphatidyl-pyridinium bisretinoid A2PE, that is the immediate precursor of A2E, a prominent constituent of RPE lipofuscin. Lipofuscin precursors such as A2PE that form in photoreceptor outer segments are phagocytosed by RPE cells upon shedding of outer segment discs. Within RPE cell lysosomes, cleavage of A2PE releases A2E and since A2E is not degraded by normal mechanisms, it accumulates. Although A2E is the best known of the RPE lipofuscin pigments, others, specifically, the all-trans-retinal dimer family of compounds, have also been isolated.. Excessive lipofuscin is believed to be toxic to the RPE by facilitating apoptosis and damage to lysosomal membranes, toxicity probably also involves the photooxidation of A2E caused by irradiation with short wavelengths of light. Consistent with this pathophysiological mechanism, an Abca4-/- abcr knockout mouse model accumulates many fold excess A2E in the RPE. Current studies in progress show preliminary, but convincing, findings that the oral administration of increased amounts of vitamin A (five times the normal dietary intake) over several months can enhance the accumulation of A2E and its precursors in the RPE cells of this animal model. It is conceivable that a similar effect could occur in patients with Stargardt disease. However, even with the extensive accumulation of lipofuscin pigments such as A2E within RPE cells, the Stargardt mouse does not show photoreceptor degeneration as noted in patients with Stargardt disease. Treatment Strategies The potential therapies listed below are based on the assumption that reducing the production of lipofuscin will slow the progression of the disease. What is currently unclear is whether a treatment strategy employed after significant accumulation of lipofuscin has already occurred will notably decrease the

3 3 lipofuscin content of the RPE cells, or will it only stabilize the amount of lipofuscin already present? Perhaps this issue needs to be more thoroughly addressed by additional pre-clinical studies prior to embarking upon human treatment trials. The treatments discussed in this section will not likely be able to restore visual function to areas of the retina that have already undergone RPE, choriocapillaris, and photoreceptor cell loss. A. Gene directed therapy Given that Stargardt disease is caused by the loss of function in ABCR protein, transfecting the wild type gene into the nucleus of photoreceptor cells could improve the retinoid flow in the visual cycle and thus avoid further accumulation of lipofuscin in RPE cells. This, in turn, could lead to a slowing (or potentially even reversing) of the disease process in patients with Stargardt disease, particularly if intervention were to occur at a sufficiently early stage of disease. Two main groups of viral vectors that have been used in gene therapy in preclinical studies are adeno-associated viruses (AAV) and lentiviruses. Both vectors have the capability to deliver genes stably and permanently into the genome of infected cells in vivo and the ability to transduce non-dividing cells. Lentiviral vectors offer the additional advantage of the ability to carry large DNA inserts such as the ABCA4 gene. At issue is whether injecting the wild type gene, within a suitable viral vector, into the subretinal space in a human patient might cause additional photoreceptor, and possibly RPE, cell damage by mechanical disruption. Further, without the ability to remove lipofuscin from the RPE cell, it is conceivable that if the RPE cells have already accumulated sufficient lipofuscin, the disease would still progress so that any further slowing of lipofuscin accumulation would not have a significant impact on a patient s disease progression. Decisions would need to be made as to the expected outcome of the gene therapy. For example, would it be to help preserve visual acuity or to reduce the progression of central atrophy and/or the enlargement of a central scotoma? B. Pharmacologic intervention The use of substances whose purpose would be to reduce the amount of available 11-cis-retinal is being explored. In a murine model, the use of fenretinide [N-(4-hydroxyphenyl)retinamide)] has shown promise in reducing the accumulation of lipofuscin. Fenretinide is a synthetic retinoid that competes with vitamin A for binding to retinol binding protein (RBP) and, as such, does not allow transthyretin to bind to this complex. The small size of the RBP-fenretinide complex allows it to be lost through the kidney s glomerular filtration. The consequence is that both RBP and vitamin A are reduced in the circulation in a dose dependent fashion. As a further consequence, there is less 11-cis-retinal

4 4 available to bind with opsin and subsequently less all-trans retinal is produced upon bleaching of visual pigment. With less all-trans retinal produced, less lipofuscin is formed in RPE cells subsequent to the shedding of outer segments. While the use of fenretinide will delay the process of recovery of rod thresholds after exposure to a bright light, it was felt that the degree of this delay would not be of likely clinical significance. Some patients may experience dry eyes and dry skin with its use. The drug s potential for teratogenic effects also needs to be considered. Inhibiting the visual cycle formation with small molecules is another potential option to prevent the formation of retinotoxic lipofuscin. The use of non-retinoid isoprenoid compounds can serve as antagonists of RPE65, a protein essential for the function of the visual cycle. These RPE65 antagonists slow the regeneration of 11-cis retinol which in turn will reduce the production of all-trans retinal and ultimately the formation of lipofuscin and A2E. The use of antioxidants is another alternative that awaits more extensive trials and pre-clinical studies. Substances that delay the development of apoptosis offer another potential option for consideration. Nevertheless, it would seem that the development of a means for reducing the level of lipofuscin within RPE cells once it has formed would be a more direct means of returning the RPE cell towards its more normal function. This approach, perhaps coupled with gene directed therapy in addition to the use of a substance that reduces the rate of lipofuscin accumulation, might offer a more long-term and successful means of managing this disease. C. Light deprivation trials Reduction of the daily light load on the retina should result in reduced production of lipofuscin since the isomerized visual chromophore all-trans-retinal is necessary for the formation of this substance. Indeed, mutant mice reared in darkness do not show abnormal accumulation of A2E and this experimental paradigm can be extrapolated to patients with monocular light deprivation with the use of black contact lenses. This approach has a theoretical advantage. Since it is a uniocular treatment, one can compare the progression in the treated vs the untreated eye, and thereby markedly reduce the sample size. While not an ultimate treatment (i.e., if the treatment were beneficial, one could not occlude both eyes of patients), a treatment effect would be a proof of principle for treatments that rely on reducing lipofuscin accumulation as their mechanism of action. Further, if occlusion is shown to slow the rate of progression, this can be used as a temporizing measure, trying to maintain the eye in a healthier state until the time that a more definitive treatment is found. The state of the disease severity when light deprivation is initiated will substantially impact upon the ultimate benefit of this or likely any forms of treatment. As a practical consideration, the actual implementation of light deprivation, particularly in children, may offer insurmountable constraints for patient compliance with

5 5 uncertain long-term benefits. The potential for the development of amblyopia in young children would also need to be considered. Patient Selection Patient selection would likely initially depend on the type of treatment strategy, (its degree of risk) and whether a phase I or phase II trial was being considered. However, the ultimate consideration is whether a treatment strategy is intended to preserve remaining visual acuity, reduce the progression of a central scotoma, or observe an improvement on an electrophysiological or psychophysical test. Data were presented on the loss of visual acuity and rate of macular pigmentary changes in patients with Stargardt disease. Patients who present within the first two decades of life will often lose visual acuity to the level of legal blindness at a substantially more rapid rate than those who present within the third decade or later. Patients who present with clinically apparent disease (foveal atrophy and flecks confined to the macula) (Fishman stage I) will most often plateau at visual acuity of 20/200 to 20/400. It is perhaps less certain whether extensive fleck lesions extending beyond the arcades are indicative of a more guarded prognosis for visual acuity loss in later years. Patients who present with no clinically evident atrophic-appearing macular lesion, as a group, have an excellent long-term prognosis for the retention of 20/25 vision or greater. There is a group of patients who present with an atrophicappearing lesion within the macula but who manifest a relative foveolar sparing, in terms of clinical appearance. As a group, such patients have a better preservation of their visual acuity. Although variability can be observed among patients, in general, when such patients show a decrease in vision to levels of 20/50, 20/60, or 20/70, they will show a subsequent further reduction in acuity to a level of 20/200 or less in a mean duration of 5.5, 3.5, and 2.0 years, respectively, a timeframe that is practical for monitoring the effect of a therapeutic intervention whose goal is to help preserve visual acuity. Data were also shown which documents that in patients with foveolar sparing, centripetal reduction in the size of the foveolar spared region, as well as centrifugal increase in the size of the perifoveal atrophic-appearing zone, can predictably be clinically observed in 3-4 years and not infrequently even at two years. This is a suitable timeframe in which to monitor the effects of treatment. One strategy for initiating a treatment trial, particularly one with minimal risk for further loss of visual function, would be to select patients with relative foveolar sparing whose visual acuity is between 20/50 and 20/70 inclusive. Likely within a period of 3-4 years, reducing the rate and/or frequency of visual loss to 20/200 or less and delaying the rate of loss of foveal structure could be monitored in a reasonable timeframe. Also, monitoring the rate of expansion at the margin of a

6 6 perifoveal zone of atrophy could also be evaluated and meaningful observations made in a period of 3-4 years. Patients could be monitored by fundus photography with subsequent digitizing of the fundus images and planimeterizing the area of fundus pigmentary change. Any changes in anatomical structure could also be monitored by autofluorescence imaging or by infrared imaging with a scanning laser ophthalmoscope. Mildly affected patients with Stargardt disease commonly show functional and structural abnormalities only at the foveal and parafoveal region, whereas severely affected patients commonly show remaining functional retina at the peripapillary region surrounding the optic nerve head. Thus the region between the fovea and the optic nerve can be used to monitor detectable changes in visual function during a treatment trial involving patients with varied severities. Such a perimetric testing strategy would require fundus visualization. The selection of patients with large central areas of both RPE and choriocapillaris atrophy within the posterior retina would likely be undesirable. Additionally, any patient with clinically apparent optic nerve pallor, suggesting atrophic changes of the optic nerve, might not be optimal for including in treatments whose focus is on improving function of the outer retina. A policy will need be developed on how to deal with patients who show a characteristic Stargardt disease clinical phenotype but no ABCA4 variants. Should such patients be considered for participation in gene based treatment trials knowing that 30-40% of ABCA4 variants/alleles cannot be identified? Monitoring a Treatment Response Various outcome measures might be utilized for documenting a potential treatment effect among which are: visual acuity, fundus perimetry, multifocal ERG, psychophysical dark-adaptation thresholds, dark-adaptation functional recovery following a bleach, fundus photography, optical coherence tomography (OCT), and fundus autofluorescence. Data were presented on the potential usefulness of fundus autofluorescence imaging for monitoring any increase in size of macular degenerative changes. At issue is whether there is a convenient, accurate, and reliable means of quantitatively measuring changes within the macula. It is not unambiguously certain that a change in autofluorescence will be associated with a change in retinal function. One can argue, however, that enlargement of a circumscribed area of loss of autofluorescence is a definite sign of progression, even if the scotoma extends past the border of the dark area. Does a reduction in initial hyper-autofluorescence during treatment establish an effectiveness for an intervention or is it conceivable that the RPE cells are further along the pathway of deterioration, which itself would also likely reduce their autofluorescence?

7 7 The use of multifocal ERG (mferg) for the measurement of cone function was felt to not likely be useful because of the inherent variability due to issues of fixation stability during testing of patients with reduced visual acuity. The use of psychophysical measurements has promise for monitoring visual function during certain treatment trials, although the issue of unsteady fixation is potentially problematic. The measurement of visual thresholds under direct visualization with the use of eye tracking and realignment programs would help to obviate the issue of poor fixation. Instruments with a suitably large operating range of stimulus intensities would be an added advantage as would the ability to determine both cone and rod thresholds. The development of commercially available perimeters with static visual field testing programs that are more specific to changes in thresholds for patients with retinal disease would be useful, but only when fixation is stable. It was suggested that monitoring of any improvement in reading speed might be of interest during a treatment trial if appropriate. Careful interpretation would be necessary to control for a learning effect. The use of a patient questionnaire as to any perceived effectiveness of treatment is of uncertain value, although its use might be considered. Other Factors to Consider in Treatment Trials Preclinical studies have provided data that suggest that light exposure and the ingestion of high doses of vitamin A could confound interpretation of results in future treatment trials. Therefore, an attempt to document light exposure, the use of sunglasses, and ingestion of vitamin A would seem prudent whenever possible. While a potentially powerful trial design would be to treat one eye and compare findings with a fellow control eye, this would require knowledge of bilateral concordance in phenotypic severity and rates of disease progression.

8 8 Summary With the discovery of mutations in the ABCA4 gene as the genetic defect in patients with autosomal recessive Stargardt disease, and a large fraction of patients with cone-rod dystrophy, a more insightful understanding of the mechanism by which RPE and photoreceptor cells degenerate in these diseases has emerged. This discovery and insight, in turn, have led to the development of an animal model of Stargardt disease and to more rational and mechanistic based treatment strategies for this disease. Currently, gene directed and pharmacologic approaches have taken center stage as potentially viable means of intervention for this disease. Independent of the treatment strategy selected, decisions need to be made as to the specific expected outcome of a treatment and particularly careful attention paid to patient selection based upon the predetermined endpoints. Paramount is whether the intent is to help preserve, delay progression, or possibly improve visual function. Will the expected outcome be to impact upon visual acuity or to affect the progression of macular atrophy and the resultant central scotoma? Regardless of the therapeutic strategy chosen, intuitively it would likely be preferable to institute monocular therapy whenever feasible. This would have the advantage of allowing a patient s other eye to serve as a control. With consideration of the above, at least for phase II or phase III studies, it would seem prudent to enroll patients with visual acuities of 20/50 to 20/70 inclusive, although this condition was fulfilled in only 21 of 82 Stargardt patients (26%) at their initial visit (J Sunness, unpublished data) and in 90 of 425 such patients (21%) (J Fishman, unpublished data). A reasonable endpoint might be the duration for loss of 3 lines in visual acuity or more on Early Treatment Diabetic Retinopathy Screening charts, which would be clinically significant. Preliminary findings suggest that the timeframe for loss to this degree would likely take place within a suitably short period of time in which to conduct a treatment trial. In addition to monitoring visual acuity change, centrifugal (inward directed) structural changes of the foveal region in a group of Stargardt patients who have relative foveolar sparing could be monitored since data are available which show that a progressive narrowing of the foveal spared region is easily observable by fundus exam and/or fundus photography in 3-4 years, and occasionally in 2 years. Further, centripetal changes, (outward expansion) of the atrophicappearing changes around the foveal region are also easily apparent in 3-4 years. These changes could be monitored by qualitative inspection but also by measurement of digitized images. Patients with 20/40 acuity or better might not be most optimal for enrollment in initial studies. Those less than 20 years of age often progress rapidly to acuity levels of 20/200 or less. Patients beyond approximately age 20 years of age with this level of acuity will often take an appreciably longer period of time to lose

9 9 substantial levels of vision. Similarly, patients with no apparent atrophicappearing macular lesion and vision of 20/25 or better will often retain their visual acuity for decades and therefore would not likely be suitable for clinical trials that use visual acuity or the development of a foveal lesion as an outcome measure. The most suitable outcome measurement(s) for future trials would likely depend upon the goal(s) of treatment as discussed above. The measurement of visual acuity, obtaining a baseline fundus examination and photographic documentation of fundus lesions would seem paramount. Additional measurements of fundus structure by the determination of fundus autofluorescence measurements would likely be of potential value, although very careful interpretation of any changes needs to be considered. The use of psychophysical thresholds could be implemented in patients considered as having steady fixation. Ideally, it would be an advantage to measure thresholds for both cone and rod function by the use of chromatic stimuli. Modification of analysis programs in currently available automated static perimetry instruments to optimize their application for patients with retinal disease would offer an added advantage for monitoring change in visual function during treatment trials. Finally, based on our current understanding of the mechanism of visual loss in patients with Stargardt disease, reducing the amount of light exposure (by the use of a visor or hat with a brim), encouraging the use of sunglasses (that optimally filter ultraviolet and blue light), in addition to not ingesting excess vitamin A (beyond the recommended daily dosage), would seem prudent. These recommendations would also likely apply to patients with other diseases in which ABCA4 mutations have been identified such as cone-rod dystrophy and a small subgroup of patients with retinitis pigmentosa.

10 10 FFB STARGARDT SYMPOSIUM ATTENDEES Friday, April 6, 2007 Coleman Center, New York, NY Rando Allikmets, Ph.D. Columbia University Dean Bok, Ph.D. University of California, Los Angeles FFB Scientific Advisory Board Diane Bovenkamp, Ph.D. Dir. of Science Information & Programs The Foundation Fighting Blindness Artur V. Cideciyan, Ph.D. University of Pennsylvania Stanley Chang, M.D. Harkness Eye Institute Columbia University Frans P.M. Cremers, Ph.D. Radbound Univ. Nijmegen Medical Centre François C. Delori, Ph.D. Harvard Medical School Gerald A. Fishman, M.D. University of Illinois FFB Scientific Advisory Board Morton F. Goldberg, M.D., F.A.C.S. Johns Hopkins Univ. School of Medicine FFB Scientific Advisory Board Carel Hoyng, Ph.D. University Hospital Nijmegen John S. Hurst, Ph.D. Grants & Awards Program Director The Foundation Fighting Blindness Richard Alan Lewis, M.D., M.S. Baylor College of Medicine Nathan L. Mata, Ph.D. Sirion Therapeutics Stephen M. Rose, Ph.D. Chief Research Office The Foundation Fighting Blindness Timothy J. Schoen, Ph.D. Director of Research Development The Foundation Fighting Blindness Benjamin Shaberman Science Writer The Foundation Fighting Blindness Paul A. Sieving, M.D., Ph.D. National Eye Institute (NEI) R. Theodore Smith, Ph.D. Columbia University Janet R. Sparrow, Ph.D. Columbia University Janet S. Sunness, M.D. Hoover Rehabilitation Services for Low Vision and Blindness Gabriel H. Travis, M.D. University of California, Los Angeles Richard G. Weleber, M.D. Oregon Health & Science University FFB Scientific Advisory Board

11 Stargardt Symposium Moving Pre-clinical Studies of Stargardt Disease into Clinical Trials Coleman Center, New York, NY Phone: Friday, April 6, :30 am 8:20 am Continental Breakfast and Coffee Introduction 8:30 am 8:45 am Welcome, Steve Rose Goals of the Symposium from Basic Scientist, Rando Allikmets Goals of the Symposium from Clinical Scientist, Gerald Fishman Session I: Molecular and Cellular Mechanisms Involved in Recessive Stargardt Disease (RSD) and Their Implication for Treatment Strategies 8:45 am 9:15 am Turnover of Lipofuscin in Retinal Pigment Epithelial Cells and Its Possible Implication for Patient Selection and Future Treatment Trials, Dean Bok 9:15 am 9:45 am Molecular and Cellular Mechanisms of Stargardt disease, Gabriel Travis 9:45 am 10:15 am Measurement of Autofluorescence as an Endpoint in Stargardt Disease, Francois Delori 10:15 am 10:45 am Anatomical Versus Functional Endpoints for RSD, Janet Sunness 10:45 am 11:00 am Coffee Break

12 12 Stargardt Symposium Page 2 Session II: Pre-Clinical Studies on Animal Models of Stargardt Disease and Their Implication for Human Treatment Trials 11:00 am 11:30 am Accumulation of RPE Lipofuscin in Retinal Pathobiology and Inhibition by Small Molecule RPE 65 Antagonists, Janet Sparrow 11:30 am 12:00 am Effects of N-(4-hydroxyphynyl) Retinamide on Vitamin A Homeostasis and A2E Biosynthesis in abcr Null Mutant Mice, Nathan Mata 12:00 am 12:30 am Correction of the Disease Phenotype in the Mouse Model of Stargardt Disease by Lentiviral Gene Therapy, Rando Allikmets 12:30 pm 1:30 pm Lunch Session III: Clinical Treatment Trials and Outcome Measures for Patients with Stargardt Disease 1:30 pm 2:00 pm Clinical Phenotypes and Progression of Visual Acuity Loss in RSD: Implications for Clinical Trials, Gerald Fishman 2:00 pm 2:30 pm Application of Psychophysical and Electrophysiological Procedures in Clinical Trials, Richard Weleber 2:30 pm 3:00 pm Quantification of Geographic Atrophy, R. Theodore Smith 3:00 pm 3:30 pm Autofluorescence as a Surrogate Endpoint and Its Correlation with Retinal Function, Artur Cideciyan 3:30 pm 5:00 pm Final Discussion and Conclusions 5:00 pm Adjourn