Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5-11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term.neurodegeneration | outer nuclear layer | retinal structure

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/ outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.retina | retinal degeneration P hotoreceptors function cooperatively with the retinal pigment epithelium (RPE) to optimize photon catch and generate signals that are transmitted to higher vision centers and perceived as a visual image. Disruption of the visual process in the retinal photoreceptors can result in blindness. Genetic defects in the retina cause substantial numbers of sight-impairing disorders by a multitude of mechanisms (1, 2). These genetic diseases were classically considered incurable, but the past few years have witnessed a new era of retinal therapeutics in which successful gene therapy of an animal model of one blinding human disease (3) was followed by stepwise translation to the clinic. The RPE65 form of Leber congenital amaurosis, due to a biochemical blockade of the retinoid cycle in the RPE, was the first and remains the only blinding genetic disease to be successfully treated in humans (reviewed in ref. 4).The next level of challenge is to initiate treatment for the majority of blinding retinal disorders in which the genetic flaws are primarily in the photoreceptors. Successful targeting of therapeutic vectors to mutant photoreceptors would be required to restore function and preserve structure. Among photoreceptor dystrophies, the X-linked forms of retinitis pigmentosa (XLRP) are one of the most common causes of severe vision loss (5). More than 25 y ago, the genetic loci were identified (6), and discovery of the underlying gene defects followed (7,8). Mutations in the reti...

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Mutations in ABCA4, which encodes a photoreceptor specific ATP-binding cassette transporter (ABCR), cause autosomal recessive forms of human blindness due to retinal degeneration (RD) including Stargardt disease. The exact disease sequence leading to photoreceptor and vision loss in ABCA4-RD is not known. Extrapolation from murine and in vitro studies predicts that two of the earliest pathophysiological features resulting from disturbed ABCR function in man would be slowed kinetics of the retinoid cycle and accelerated deposition of lipofuscin in the retinal pigment epithelium (RPE). To determine the human pathogenetic sequence, we studied surrogate measures of retinoid cycle kinetics, lipofuscin accumulation, and rod and cone photoreceptor and RPE loss in ABCA4-RD patients with a wide spectrum of disease severities. There were different extents of photoreceptor/RPE loss and lipofuscin accumulation in different regions of the retina. Slowing of retinoid cycle kinetics was not present in all patients; when present, it was not homogeneous across the retina; and the extent of slowing correlated well with the degree of degeneration. The orderly relationship between these phenotypic features permitted the development of a model of disease sequence in ABCA4-RD. The model predicted lipofuscin accumulation as a key and early component of the disease expression in man, as in mice. In man, however, abnormal slowing of the rod and cone retinoid cycle occurs at later stages of the disease sequence. Knowledge of the human ABCA4 disease sequence will be critical for defining rates of progression, selecting appropriate patients and retinal locations for future therapy, and choosing appropriate treatment outcomes.

Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.

Autosomal recessive retinal diseases caused by mutations in the ABCA4 gene are being considered for gene replacement therapy. All individuals with ABCA4-disease show macular degeneration, but only some are thought to progress to retina-wide blindness. It is currently not predictable if or when specific ABCA4 genotypes will show extramacular disease, and how fast it will progress thereafter. Early clinical trials of focal subretinal gene therapy will aim to arrest disease progression in the extramacular retina. In 66 individuals with known disease-causing ABCA4 alleles, we defined retina-wide disease expression by measuring rod- and cone-photoreceptor-mediated vision. Serial measurements over a mean period of 8.7 years were consistent with a model wherein a normal plateau phase of variable length was followed by initiation of retina-wide disease that progressed exponentially. Once initiated, the mean rate of disease progression was 1.1 log/decade for rods and 0.45 log/decade for cones. Spatio-temporal progression of disease could be described as the sum of two components, one with a central-to-peripheral gradient and the other with a uniform retina-wide pattern. Estimates of the age of disease initiation were used as a severity metric and contributions made by each ABCA4 allele were predicted. One-third of the non-truncating alleles were found to cause more severe disease than premature truncations supporting the existence of a pathogenic component beyond simple loss of function. Genotype-based inclusion/exclusion criteria and prediction of the age of retina-wide disease initiation will be invaluable for selecting appropriate candidates for clinical trials in ABCA4 disease.

The health of the retinal pigment epithelium (RPE) can be estimated with autofluorescence (AF) imaging of lipofuscin, which accumulates as a byproduct of retinal exposure to light. Lipofuscin may be toxic to the RPE, and its toxicity may be enhanced by short-wavelength (SW) illumination. The high-intensity and SW excitation light used in conventional AF imaging could, at least in principle, increase the rate of lipofuscin accumulation and/or increase its toxicity. We considered two reduced-illuminance AF imaging (RAFI) methods as alternatives to conventional AF imaging. RAFI methods use either near-infrared (NIR) light or reduced-radiance SW illumination for excitation of fluorophores. We quantified the distribution of RAFI signals in relation to retinal structure and function in patients with the prototypical lipofuscin accumulation disease caused by mutations in ABCA4. There was evidence for two subclinical stages of macular ABCA4 disease involving hyperautofluorescence of both SW- and NIR-RAFI with and without associated loss of visual function. Use of RAFI methods and microperimetry in future clinical trials involving lipofuscinopathies should allow quantification of subclinical disease expression and progression without subjecting the diseased retina/RPE to undue light exposure.

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.