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THE murine retinal degeneration slow (rds) gene is a semidominant mutation with a phenotype having rod and cone photoreceptors that develop abnormally and then slowly degenerate1–3. The phenotype is a possible model for retinitis pigmentosa, one of the scores of hereditary human retinal degenerations, which is also characterized by photoreceptor degeneration. We report here three mutations of the human homologue of the rds gene (RDS) that cosegregate with autosomal dominant retinitis pigmentosa in separate families. Our results indicate that some cases of autosomal dominant retinitis pigmentosa are due to mutations at the RDS locus.

The inherited outer retinal dystrophies comprise a large number of disorders characterised by a slow and progressive retinal degeneration. They have been arbitrarily divided into macular dystrophies, retinitis pigmentosa (RP), and cone/rod dystrophies on the basis of their phenotype. They are the result of mutations in genes that are presumed to express in either the photoreceptor cells or the retinal pigment epithelium.1 Retinitis pigmentosa is believed to affect about one in 3500 of the population,2and the prevalence of remaining dystrophies is probably similar. The mode of inheritance can be autosomal dominant, autosomal recessive, X linked recessive,3 or digenic.4 5 With few exceptions such as α,β lipoproteinaemia6 7and Refsum disease,8 there is no treatment by which the primary disorder can be modified. Some symptomatic relief may be derived from cataract extraction,9 and carbonic anhydrase inhibition if there is macular oedema.10 Vitamin A supplementation may slow the progression although this has been the subject of controversy.11 12 Although there is no treatment by which the primary disorder can be manipulated, the patients can expect a reasonable account of visual prognosis, accurate genetic counselling, and support for rehabilitation. In addition, those affected are also keen to know the progress in research for a cure particularly as they have become progressively more aware of the potential for treatment through the various self help associations such as the British Retinitis Pigmentosa Society, the internet, and the press. Researchers around the world are working on different potential therapeutic approaches to the problem. These are based on the possibility of modulation of cell death by growth factors, transfecting the photoreceptor or retinal pigment epithelial cell with functioning genes, transplanting photoreceptor or retinal pigment epithelial cells into the subretinal space, or the use of electronic devices to …

The photoreceptor degeneration seen in age-related macular degeneration (AMD) and retinitis pigmentosa (RP) represents the major cause of blindness in developed countries, for which no cure is available.1–3 Cell-based therapies have been shown effective in rescuing vision in animal models of retinal degeneration.4–9 For donor cells to be suitable in a clinical setting they should be human derived, must be effective in reversing or slowing the degenerative events, must be readily renewable and not senescent, and must be effective over a long period. In a series of studies, we have used the Royal College of Surgeons (RCS) rat to explore the efficacy of cell-based therapies in rescuing vision. In this animal, the retinal pigment epithelial cell (RPE) fails to phagocytose shed outer segment material at a normal rate because of a mutation in the Mertk gene.10 This results in an accumulation of outer segment debris and subsequently leads to photoreceptor cell loss.11,12 Although comparable defects have been seen in a cohort of patients with retinitis pigmentosa,13 the animal does serve to some degree as a model for photoreceptor loss caused by defective or dysfunctional RPE and for retinitis pigmentosa in general. In previous work,14 we showed that human cortical neural progenitor cells (hNPCctx) rescued vision at near normal levels when injected into the subretinal space of the RCS rat. Some-what unexpectedly, they formed an RPE-like layer between photoreceptors and the host RPE layer and migrated to the retina. Given the potential for such cells to provide a therapy for degenerative diseases throughout the central nervous system,15 including the retina, we felt it important to evaluate the long-term effects of grafting. Critical questions that remained centered on the duration of cell survival and vision rescue and on the long-term behavior of and host response to the transplanted cells. To answer these questions, we observed a new group of rats that underwent transplantation at postnatal day (P) 21; functional tests were conducted at approximately P90, P150, and P280. A crucial part of the study was to observe the performance of individual rats at three times. Some rats were observed separately for validation of the previous morphologic results at the earlier times and to provide a context for the later data.

Early-onset, severe retinal dystrophy caused by mutations in the gene encoding retinal pigment epithelium–specific 65-kD protein (RPE65) is associated with poor vision at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the three patients. We detected no change in retinal responses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747.), Supported by grants from the U.K. Department of Health, the British Retinitis Pigmentosa Society, and the Special Trustees of Moorfields Eye Hospital, and by the Sir Jules Thorn Charitable Trust, the Wellcome Trust, the European Union (EVI-Genoret and Clinigene programs), the Medical Research Council, Foundation Fighting Blindness, Fight for Sight, the Ulverscroft Foundation, Fighting Blindness (Ireland), Moorfields Eye Hospital, and Institute of Ophthalmology Biomedical Research Centre for Ophthalmology, University College London.

Preface.- About The Editors.- Travel Awardees.- Part I Keynote Lectures.- Curing Blindness With Stem Cells: Hope, Reality, And Challenges.- Retinal Degenerations: Planning For The Future.- Part Ii Neuroprotection.- Neurotrophins Induce Neuroprotective Signaling In The Retinal Pigment Epithelial Cell By Activating The Synthesis Of The Anti-Inflammatory And Anti-Apoptotic Neuroprotectin D1.- On The Role Of Cntf As A Potential Therapy For Retinal Degeneration.- Nanoceria Particles Prevent Roi-Induced Blindness.- An In-Vivo Assay To Identify Compounds Protective Against Light Induced Apoptosis.- Role Of Bcl-Xl In Photoreceptor Survival.- The Hypoxic Transcriptome Of The Retina.- Part Iii Gene Therapy And Neuroprotection.- Lentiviral Gene Transfer-Mediated Cone Vision Restoration In Rpe65 Knockout Mice.- In Vitro Analysis Of Ribozyme-Mediated Knockdown Of An Adrp Associated Rhodopsin Mutation.- Gene Therapy For Mouse Models Of Adrp.- Development Of Viral Vectors With Optimal Transgene Expression For Ocular Gene Therapies.- Adeno-Associated Viral Vectors And The Retina.- Genetic Supplementation Of Rds Alleviates A Loss-Of-Function Phenotype In C214s Model Of Retinitis Pigmentosa.- Morphological Aspects Related To Long-Term Functional Improvement Of The Retina In The 4 Years Following Raav-Mediated Gene Transfer In The Rpe65 Null Mutation Dog.- Virus-Mediated Gene Delivery To Neuronal Progenitors.- Part Iv Animal Models Of Retinal Degeneration.- Loss Of Visual And Retinal Function In Light-Stressed Mice.- Erg Responses And Microarray Analysis Of Gene Expression In A Multifactorial Murine Model Of Age-Related Retinal Degeneration.- Oxygen Supply And Retinal Function: Insights From A Transgenic Animal Model.- Characterization Of Gene Expression Profiles Of Normal Canine Retina And Brain Using A Retinal Cdna Microarray.- Toward A Higher Fidelity Model Of Amd.- The Potential Of Ambient Light Restriction To Restore Function To The Dengerating P23h-3 Rat Retina.- Part V Molecular Genetics And Candidate Genes.- Mutations In Known Genes Account For 58% Of Autosomal Dominant Retinitis Pigmentosa (Adrp).- Genetics Of Age-Related Macular Degeneration.- Retinal Phenotype Of An X-Linked Pseudo-Usher Syndrome In Association With The G173r Mutation In The Rpgr Gene.- Mutation In The Pyk2-Binding Domain Of Pitpnm3 Causes Autosomal Dominant Cone Dystrophy (Cord5) In Two Swedish Families.- Identification And Characterization Of Genes Expressed In Cone Photoreceptors.- Clinical And Genetic Characterization Of A Chinese Family With Csnb1.- 10q26 Is Associated With Increased Risk Of Age-Related Macular Degeneration In The Utah Population.- Part Vi Diagnostic, Clinical, Cytopathological And Physiologic Aspects Of Retinal Degeneration.- Carboxyethylpyrrole Adducts, Age-Related Macular Degeneration And Neovascularization.- A Possible Impaired Signaling Mechanism In Human Retinal Pigment Epithelial Cells From Patients With Macular Degeneration.- Expression and Cell Compartmentalization of Efemp1, A Protein Associated with Malattia Leventinese.- Role of Elovl4 in Fatty Acid Metabolism.- Organization and Molecular Interactions of Retinoschisin in Photoreceptors.- Part VII Basic Science Underlying Retinal Degeneration.- Proteomics Profiling of the Cone Photoreceptor Cell Line, 661W.- G -Secretase Regulates Vegfr-1 Signalling in Vascular Endothelium and RPE.- Analysis of the Rate of Disk Membrane Digestion by Cultured RPE Cells.- Functional Expression of Cone Cyclic Nucleotide-Gated Channel in Cone Photoreceptor-Derived 661W Cells.- Phosphorylation of Caveolin-1 in Bovine Rod Outer Segments in Vitro by an Edogenous Tyrosine Kinase.- Regulation of Neurotrophin Expression and Activity in the Retina.- Involvement of Guanylate Cyclases in Transport of Photoreceptor Peripheral Membrane Proteins.- Rod Progenitor Cells in the Mature Zebrafish Retina.- Avss5 Integrin Receptors at the Apical Surface of The RPE: One Receptor, Two Functions.- Implantation of Mouse Eyes

Retinitis pigmentosa (RP) is the name given to a group of eye diseases often characterized by night blindness and the gradual loss of peripheral vision RP causes the degeneration of photoreceptor cells in the retina. As these cells degenerate and die, patients experience progressive vision loss to eventual blindness. The most common feature of all forms of RP is a gradual degeneration of photoreceptor cells: rod cells and cone cells. The rods and cones are the cells responsible for converting light into electrical impulses that are transmitted to the brain where “seeing” actually occurs. RP is an inherited, genetically heterogeneous condition, i.e., RP can result from mutations in many different genes. It is caused by mutations in genes that are active in retinal cells. RP is a common form of retinal degeneration (RD). Mouse models of RD have been investigated for many years to understand the causes of photoreceptor cell death. Knowledge about these mutations is essential for proper selection of mouse models for use in research. In this chapter, we review these naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types. The mutations are described in chronological order and provide a list of the mouse strains that carry each mutation.

A hereditary degenerative disease of the retina, retinitis pigmentosa (RP), is the leading cause of visual handicap among working populations in developed countries, with an estimated 1.5 million patients worldwide (1,2). Clinically, RP is characterized by night blindness (nyctalopia) as a result of initial death of rod photoreceptors, followed by progressive visual loss owing to secondary degeneration of cone cells (3). Mutations underlying RP reveal a genetically heterogenous condition, which can be inherited in an autosomal dominant (AD), recessive, X-linked recessive, digenic, or mitochondrial mode, with around 40 known or predicted genes implicated in disease pathology (http://www.sph.uth.tmc.edu/RetNet/). Of those genes with known functions, some encode proteins involved in the visual transduction cascade, e.g., rhodopsin, others in maintenance of photoreceptor structure, e.g., peripherin, and others involved in regeneration of the rhodopsin chromophore (11-cis-retinal) in the visual cycle, e.g., retinal pigment epithelial protein (RPE) 65 (4). Interestingly, a number of RP genes are widely expressed but only cause disease pathology within the retina, highlighting the unique and complex biochemistry of photoreceptor cells. Included in the latter category are the genes HPRP3 and PRPC8 encoding pre-messenger RNA splicing factors and the gene encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), the rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis (5, 6, 7). Despite such genetic heterogeneity, photoreceptors degenerate in RP, and indeed in other inherited retinal degenerations, by a common form of cell death, apoptosis (8,9).

Ribozymes targeted to a point mutation (histidine for proline substitution at position 23) in rhodopsin were evaluated as therapeutic agents for photoreceptor degeneration in an animal model of autosomal dominant retinitis pigmentosa (ADRP). Recombinant adeno-associated virus (AAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. AAV was administered by subretinal injection at postnatal day (P) 15 to rats expressing a rod opsin transgene with a proline-to-histidine substitution in exon 1 at codon 23 of the rhodopsin gene (P23H). In vivo levels of normal and mutant RNAs were measured by allele-specific RT-PCR at P60. Eyes were examined by histopathology and morphometric analysis, as well as by ERG at approximately P60, P75 or P90. Expression of either a hammerhead or hairpin ribozyme markedly slowed the rate of photoreceptor degeneration for at least 3 months as determined by outer nuclear layer thickness as well as by inner and outer segment length. Catalytically inactive control ribozymes had a significantly smaller effect on the retinal degeneration. Minimal rescue effects were seen in retinas injected with either AAV containing a gene for green fluorescent protein rather than the ribozyme gene or buffer only. The level of mutant opsin RNA relative to wild-type RNA was also reproducibly lower in ribozyme-treated retina. Finally, ERG comparisons of ribozyme-treated versus control eyes demonstrated functional rescue. We conclude that ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapy for autosomal dominant retinal degeneration.

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal blinding diseases, such as Leber congenital amaurosis and retinitis pigmentosa. In the previous studies we have shown that Crb1 resides in retinal Muller glia cells and that loss of Crb1 results in retinal degeneration (particularly in the inferior temporal quadrant of the mouse eye). Degeneration is increased by exposure to white light. Here, we studied the role of light and aging to gain a better understanding of the factors involved in the progress of retinal disease. Our data reveal that light is neither sufficient nor required to induce retinal disorganization and degeneration in young Crb1(-/-) mutant mice, suggesting that it rather modulates the retinal phenotype. Gene expression profiling showed that expression of five genes is altered in light-exposed Crb1(-/-) mutant retinas. Three of the five genes are involved in chromosome stabilization (Pituitary tumor transforming gene 1 or Pttg1, Establishment of cohesion 1 homolog 1 or Esco1, and a gene similar to histone H2B). In aged retinas, degeneration of photoreceptors, inner retinal neurons, and retinal pigment epithelium was practically limited to the inferior temporal quadrant. Loss of Crb1 in Muller glia cells resulted in an irregular number and size of their apical villi. We propose that Crb1 is required to regulate number and size of these Muller glia cell villi. The subsequent loss of retinal integrity resulted in neovascularization, in which blood vessels of the choroid protruded into the neural retina.

Background RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA). Methods and Findings RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation. Conclusions Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease., The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients., Editors' Summary Background. The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time. Why Was This Study Done? Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision. What Did the Researchers Do and Find? The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals. What Do These Findings Mean? The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040230. General information on gene therapy is available from the Oak Ridge National Laboratory Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation) The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis ClinicalTrials.gov gives details on treatment trials for Leber congenital amaurosis The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud) The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews WonderBaby, information for parents of babies with Leber congenital amaurosis

Retinitis pigmentosa is a genetically heterogeneous group of progressive retinal degenerations.1 Autosomal recessive retinitis pigmentosa caused by mutations in the gene encoding the β-subunit of rod photoreceptor cyclic guanosine monophosphate-phosphodiesterase (PDE6B) was one of the first forms to be identified, and there are well-studied murine and canine animal models as well as proof-of-concept success of somatic gene therapy.1–4 Rapid rod photoreceptor degeneration in the animal models is complicated by morphological changes involving the inner retina.5,6 It is unknown, however, whether the human form of retinitis pigmentosa is also complicated by retinal remodelling; the answer could have implications for treatment potential. We used optical coherence tomography (OCT) to study the retina of a patient with retinitis pigmentosa with a known PDE6B null mutation,7 and found there was abnormal laminar architecture suggesting retinal remodelling. A 25-year-old woman with retinitis pigmentosa was homozygous for the Cys270X mutation in PDE6B . There was no rod function and only severely impaired cone function.7 At 34 years of age, …

The purpose of this study was to explore neuroretinal transplantation in a large animal model of severe retinitis pigmentosa and to establish graft development, long-term survival, graft-host integration, and effects on the host retina. Methods Rhodopsin transgenic pigs, aged 6 months, received in one eye a fetal full-thickness neuroretinal sheet in the subretinal space by means of vitrectomy and retinotomy. Six months postoperatively, eyes were studied in the light microscope and with immunohistochemical markers. Full-field electroretinography (ERG) was performed at 4 and 6 months. Results Laminated grafts with well-organized photoreceptors, rod bipolar cells, and Muller cells were found in five of six eyes. Neuronal connections between graft and host retina were not seen. In the five eyes containing a graft, the number of surviving rods in the host retina was significantly higher compared with unoperated eyes. The ERG did not reveal any significant difference in b-wave amplitude between operated and control eyes, but the cone-derived response in operated eyes increased significantly from 4 to 6 months while the rod response in control eyes decreased significantly. Conclusions Fetal full-thickness neuroretina can be transplanted safely to an eye with severe retinal degeneration. In their major part, the transplants develop a normal laminated morphology and survive for at least 6 months. Graft and host retinal neurons do not form connections. Retinal function in the host is reduced initially by the surgical trauma, but the presence of a well-laminated graft counteracts this effect and rescues rods from degeneration.

Fibulin-5 is an extracellular matrix glycoprotein that participates in elastogenesis. Mutations in the gene for fibulin-5 have been found to be associated with age-related macular degeneration. Little is known, however, about the expression of this gene in normal eyes or eyes with age-related macular degeneration. In this study, we evaluated the expression of the fibulin-5 protein in human donor eyes and localized this protein to Bruch's membrane and the intercapillary pillars of the choriocapillaris in normal eyes. In eyes with age-related macular degeneration, fibulin-5 was localized to pathologic basal deposits beneath the retinal pigment epithelium as well as some small drusen. These results suggest that fibulin-5 may promote extracellular deposit formation in macular degeneration.

Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse.For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections.Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected.Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Purpose To characterize the course of retinal disease in X-linked progressive retinal atrophy 2 (XLPRA2), a canine model of early onset X-linked retinitis pigmentosa (XLRP) caused by a two-nucleotide microdeletion in RPGR ORF15. Methods The retinas of 25 XLPRA2-affected dogs (age range, 2-40.6 weeks) and age-matched control subjects were collected, fixed, and embedded in epoxy resin for morphologic evaluation or in optimal cutting temperature (OCT) medium for TUNEL assay and immunohistochemistry. Cell-specific antibodies were used to examine changes in rods and cones and to evaluate the effects of the primary photoreceptor degeneration on inner retinal cells. Results Abnormal development of photoreceptors was recognizable as early as 3.9 weeks of age. Outer segment (OS) misalignment was followed by their disorganization and fragmentation. Reduction in length and broadening of rod and cone inner segments (IS) was next observed, followed by the focal loss of rod and cone IS at later time points. The proportion of dying photoreceptors peaked at approximately 6 to 7 weeks of age and was significantly reduced after 12 weeks. In addition to rod and cone opsin mislocalization, there was early rod neurite sprouting, retraction of rod bipolar cell dendrites, and increased Muller cell reactivity. Later in the course of the disease, changes were also noted in horizontal cells and amacrine cells. Conclusions XLPRA2 is an early-onset model of XLRP that is morphologically characterized by abnormal photoreceptor maturation followed by progressive rod-cone degeneration and early inner retina remodeling. The results suggest that therapeutic strategies for this retinal degeneration should target not solely photoreceptor cells but also inner retinal neurons.

Animal models are powerful tools with which to investigate the aetiology of human retinal degenerations, especially retinitis pigmentosa (RP) and allied diseases. Gene defects identified in some animal models were later discovered in orthologous genes in human RP families. Keywords: retinitis pigmentosa; animal models; transgenes; gene knockout; retinal degeneration; phototransduction; visual cycle

Inherited retinal degenerations are a group of clinically and genetically heterogeneous diseases characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Mutations in the rhodopsin gene are common causes of autosomal dominant retinitis pigmentosa (RP). Among them the P23H amino-acid substitution represents the most frequent rhodopsin mutation in US. Given the gain of function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable.

Leber congenital amaurosis (LCA) is a retinitis pigmentosa with early onset, leading to blindness in infants. There is currently no efficient therapy to treat LCA. At the present time, mutations in seven different genes have been associated with the disease (Hanein et al. 2004). In 10 to 15% of the cases LCA originates from a mutation in RPE65 (Gu et al. 1997), a gene specifically expressed in the cells of the retinal pigment epithelium layer (RPE cells). This gene encodes a 65kD protein the function of which has been dissected in a recently published study demonstrating its crucial role as a regulator of the visual cycle and a chaperone for the chromophore of the visual pigment (Xue et al. 2004). The patients affected by a mutation in this gene could benefit from a substitutive gene therapy consisting in the transfer of a fully functional allele of the RPE65 gene in RPE cells. Furthermore, animal models of RPE65 mutations have been identified (Aguirre et al. 1998; Veske et al. 1999) or genetically produced (Redmond et al. 1998) and thus provide the necessary tools to set up the conditions of such a strategy before a clinical trial can be started. The proof of feasibility of this approach has indeed already been established in dogs bearing a spontaneous mutation in the RPE65 gene (Acland et al. 2001; NarfstrOm et al. 2003), as well as in knock-out mice (Dejneka et al. 2004; Lai et al. 2004). These studies have shown that an adeno-associated virus (AAV)-derived vector is able to deliver the RPE65 gene to RPE cells and thus to restore vision at least partially. Nevertheless, before a clinical trial can take place, a great effort must be provided to assess the bio-safety of the procedure. In particular, trans-gene expression has to be tightly controlled to achieve the following criteria: (i) expression should occur only in RPE cells; (ii) expression should reach the therapeutic level without disturbing the homeostasis of the target cells.

Vectors based on the adeno-associated virus (rAAV) are able to transduce the retina of animal models, including non-human primates, for a long-term period, safely and at sustained levels. The ability of the various rAAV serotypes to transduce retinal target cells has been exploited to successfully transfer genes to photoreceptors, retinal pigment epithelium and the inner retina, which are affected in many inherited and non-inherited blinding diseases. rAAV-mediated, constitutive and regulated gene expression at therapeutic levels has been achieved in the retina of animal models, thus providing proof-of-principle of gene therapy efficacy and safety in models of dominant and recessive retinal disorders. In addition, gene transfer of molecules with either neurotrophic or antiangiogenic properties provides useful alternatives to the classic gene replacement for treatment of both mendelian and complex traits affecting the retina. Years of successful rAAV-mediated gene transfer to the retina have resulted in restoration of vision in dogs affected with congenital blindness. This has paved the way to the first attempts at treating inherited retinal diseases in humans with rAAV. Although the results of rAAV clinical trials for non-retinal diseases give a warning that the outcome of viral-mediated gene transfer in humans may be different from that predicted based on results in other species, the immune privilege of the retina combined with the versatility of rAAV serotypes may ultimately provide the first successful treatment of human inherited diseases using rAAV.

Retinal pigment epithelium (RPE) arises from neuroectoderm and plays a key role in support of photoreceptor functions. Several degenerative eye diseases, such as macular degeneration or retinitis pigmentosa, are associated with impaired RPE function that may lead to photoreceptor loss and blindness. RPE derived from human embryonic stem (hES) cells can be an important source of this tissue for transplantation to cure such degenerative diseases. This chapter describes differentiation of hES cells to RPE, its subsequent isolation, maintenance in culture, and characterization.

Gene transfer is a promising approach for single gene defects occurring in several retinal degenerations, such as Leber congenital amaurosis (LCA), a group of severe retinal dystrophies affecting children. About 10% of LCA cases are due to a mutation in RPE65, a key enzyme for the metabolism of Vitamin A during the visual cycle. The consequence of RPE65 deficiency is a lack of the 11-cis- retinal chromophore at the photoreceptor level and an associated retinal degeneration. Gene therapy in dogs lacking RPE65 protein restores visual function on the long term (Acland et al., Nat Genet 28: 92; Narfstrom et al., Invest Ophthalmol Vis Sci 44: 1663). However no confirmed indications on cone rescue and cone function restoration, necessary for day vision and high visual acuity, have been reported in the different animal models used in gene therapy studies. To evaluate the efficiency of Rpe65 replacement by gene transfer in rodent models of LCA, we have constructed a lentiviral vector expressing the Rpe65 mouse cDNA under the control of a promoter fragment from the human RPE65 gene. We studied the therapeutic efficiency of this vector in the Rpe65 knockout mouse as well as in the Rpe65/Gnat1a double knockout mouse, to reveal the effect of Rpe65 gene transfer on the cone photoreceptors. Gnat1a-/- mice do not have any rod function. Thus, any photoreceptor activity occurring in the Rpe65/Gnat1a double knockout background after Rpe65 gene replacement should arise from cone photoreceptors.

Background RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. Methods and Findings Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. Conclusions By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65 −/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose., In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function., Editors' Summary Background. Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves. Why Was This Study Done? Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed. What Did the Researchers Do and Find? Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice. What Do These Findings Mean? These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347. The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis

Retinal dystrophies are a common cause of blindness in purebred dogs. Progressive retinal atrophy, the canine equivalent of retinitis pigmentosa in humans, is the most common dystrophy. Molecular studies have led to the identification of the genetic defect underlying some forms of progressive retinal atrophy and the mapping of the chromosomal location of others. Additionally, the gene mutation that causes a severe retinal dystrophy in the briard, which is the equivalent of Leber congenital amaurosis in humans, has been identified. These advances have led to the development of DNA-based diagnostic tests for some retinal dystrophies, thus facilitating their eradication. The study of these dystrophies in dogs has also provided useful information about the equivalent diseases in humans. Recently, gene therapy has been used to restore vision to dogs with a retinal dystrophy due to a mutation in the RPE65 gene. Such studies are important in the quest to develop therapies for similar conditions in humans.

Many systemic and eye-specific genetic disorders are accompanied by retinal degenerations that lead to blindness. In some of these diseases retinal degeneration occurs early in life and is quite rapid, whereas in other disorders, retinal degeneration starts later and progresses very slowly. At present, no therapies are available to patients for preventing or reversing the retinal degeneration that occurs in these diseases. Implantation of neural progenitor cells into the eye may be a means by which to retard or even reverse degeneration of the retina. To evaluate the potential of neural precursor cell implantation for treating retinal degenerative disorders, neuralized mouse embryonic stem cells from green fluorescent protein (GFP) transgenic mice were administered intravitreally to normal mice, mice with early retinal degeneration, and mice with slowly progressing retinal degeneration. In normal mice, the donor cells remained in the vitreous cavity and did not associate with the host retina. In mice with early retinal degeneration, implantation of the neural precursors was performed after the degeneration was almost complete. In these animals, the donor cells primarily associated closely with the inner surface of the retina, although a small fraction of donor cells did integrate into the host retina. Donor cells implanted in mice with slowly progressing retinal degeneration also associated with the inner retinal surface, but many more of the cells integrated into the retina. These findings indicate the importance of host tissue-donor cell interactions in determining the fate of implanted neural precursor cells. These interactions will be a major consideration when devising strategies for using cell implantation therapies for neurodegenerative disorders.

Mutations in RPE65 , a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65 -mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65 -mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65 -/- mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Several retinal dystrophies, including retinitis pigmentosa type 12 and Leber congenital amaurosis, are caused by a large variety of mutations in the CRB1 (Crumbs homologue 1) gene. This discovery led to an increased focus on the function of CRB1 and the Drosophila homologue Crumbs. In the present study, we review the current knowledge on Crumbs and its vertebrate homologues, their function in cell polarity and their pathogenicity in retinal degeneration.

Leber congenital amaurosis (LCA) is the most severe inherited retinopathy, with the earliest age of onset. Because this currently incurable disease is present from birth and is a relatively rare disorder, the development of animal models that closely resemble the phenotype in patients is especially important. Our previous genetic analyses of LCA patients identified mutations in the aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene. Here we present development of an animal model of AIPL1-associated LCA, the Aipl1-deficient mouse. Aipl1 is expressed at low levels throughout human and mouse retinal development and is rapidly upregulated as photoreceptors differentiate. The mouse displays rapid retinal degeneration and massive Muller cell gliosis, resembling the phenotype of the rd mouse, which is caused by a mutation in the gene for the β-subunit of the rod-specific phosphodiesterase. We confirm that this phenotype is consistent with the human disease using electroretinograms, and document the disease pathogenesis by analyzing the development of all retinal cell types and synaptogenesis during retinal histogenesis. Ectopic expression of AIPL1 led to deregulated retinal progenitor cell proliferation and alterations in cell fate specification; however, no gross abnormalities of proliferation during retinal development were detected. Data from analysis of proliferation and cell fate specification during retinal development of Aipl1-deficient mice suggests that there may be redundancy or compensation for Aipl1 loss by other related proteins. Because this mouse model closely mimics the human retinopathy caused by homozygous mutations in this gene, it provides a preclinical model for testing therapies to rescue the vision of children whose blindness is caused by AIPL1 mutations.

Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow–derived stem cells (lineage-negative hematopoietic stem cells [Lin– HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin– HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.

Leber's congenital amaurosis (LCA) is the earliest and most severe form in the world of genetic retinal dystrophy causing blindness. An animal model of LCA was recently created in which the cone-rod homeobox (crx) gene was disrupted using homologous recombination. Crx-/- mice display abnormal development of photoreceptors followed by their degeneration. We analyzed the morphology of inner retinal cells in crx-/- mice in order to evaluate the effects of abnormal photoreceptor development and death upon other retinal neurons. The identification of a time window during which inner retinal cells are still viable could be very important in view of the possibilities that photoreceptor transplantation or gene therapy might be used to restore vision in LCA. We used a combination of immunocytochemical and confocal microscopy techniques to screen the crx-/- inner retina and verify its morphological integrity after photoreceptor degeneration. We found significant morphological alterations in second-order neurons in crx-/- animals. The appearance of mutant retinas after photoreceptor death is indistinguishable from that of the retinal degeneration (rd/rd) mouse, a different genetic model of a retinal disease characterized by photoreceptor degeneration. However, at early stages of photoreceptor degeneration the morphology of retinal cells in the crx-/- mutant is considerably well preserved. It is likely that different genetic mechanisms that cause abnormal photoreceptor development and/or degeneration lead to a common pathway that determines second-order neuron modifications. The severity of modifications is linked to the timing of onset of the degeneration and appears to increase with time. (C) 2004 Wiley-Liss, Inc.

Retinitis pigmentosa (RP) is a leading cause of human blindness due to degeneration of retinal photoreceptor cells. Causes of retinal degeneration include defects in the visual pigment, defects in the proteins important for photoreceptor function or in enzymes involved in initiating visual transduction. Despite the diversity of genetic mutations identified in inherited forms of retinal dystrophy, there is a common end result of photoreceptor death and functional blindness. In this review, pertinent anatomical and physiological pathways involved in RP and the underlying genetic mutations are outlined, including a discussion on the inheritance patterns revealed by advances in molecular biological techniques. Characteristics of progression rates of visual field loss and current management options will provide useful clinical guidelines for the management of patients with RP.

The congenital retinal blindness known as Leber congenital amaurosis (LCA) can be caused by mutations in the RPE65 gene. RPE65 plays a critical role in the visual cycle that produces the photosensitive pigment rhodopsin. Recent evidence from human studies of LCA indicates that earlier rather than later intervention may be more likely to restore vision. We determined the impact of in utero delivery of the human RPE65 cDNA to retinal pigment epithelium cells in a murine model of LCA, the Rpe65(-/-) mouse, using a serotype 2 adeno-associated virus packaged within an AAV1 capsid (AAV2/1). Delivery of AAV2/1-CMV-hRPE65 to fetuses (embryonic day 14) resulted in efficient transduction of retinal pigment epithelium, restoration of visual function, and measurable rhodopsin. The results demonstrate AAV-mediated correction of the deficit and suggest that in utero retinal gene delivery may be a useful approach for treating a variety of blinding congenital retinal diseases.

Purpose To evaluate the retina in autopsy eyes from patients over age 60 with autosomal dominant retinitis pigmentosa and a mutation in the RP13 gene (designated as PRPC8, Arg2310Gly), rhodopsin Pro23His, rhodopsin Cys110Arg, or rhodopsin Glu181Lys. Design Histologic study of the retina. Methods All eyes were prepared for electron microscopy within 12 hours after death. Results All eyes showed loss of rod photoreceptors. Remaining cones showed perinuclear membranous swirls, inclusion bodies in the inner segments, and shortened or absent outer segments despite causation by various gene defects. Conclusion The comparable histologic findings in these four cases suggest a final common pathway leading to photoreceptor cell death in these dominant forms of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disorder clinically characterized by a pale, waxy optic nerve head, attenuated retinal blood vessels and bone spicule pigment in the retina. Hemodynamic studies have demonstrated that RP is associated with a reduction in the retinal and choroidal blood flow. Retinal hemodynamic impairment is also present in the early stages of RP and various hypotheses have been advanced as to its cause. The authors studied 20 patients, 12 males and 8 females, aged between 26 and 42 years (mean 34.6 years) affected by simplex RP. The twenty patients were divided in two groups according to the degree of sight impairment: group A consisted of 10 patients with a visual acuity of 0.3 +/- 0.1, visual field mean defect 18.988 +/- 3.419 dB and b-wave electroretinogram amplitude of 13.14 +/- 0.308 microV. Group B consisted of 10 patients with a visual acuity of 0.8 +/- 0.2, visual field MD 10.523 +/- 3.582 dB and b-wave electroretinogram amplitude of 26.000 +/- 0.757 microV. An increase in plasma levels of endothelin-1 (ET-1) was found as compared with healthy controls: 1.910 +/- 0.617 pg/ml vs. 1.180 +/- 0.210 pg/ml (p < 0.021), but there was no statistical difference between group A and B (p < 0.163). It is thought that an increase in ET-1 and retinal oxygen levels in RP could lead to vasoconstriction and a decrease in the retinal blood flow worsening the abiotrophic process.

Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin ( RHO ) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the RHO gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with RHO mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in RHO mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The RHO mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies.

Leber congenital amaurosis (LCA) is the most severe inherited retinal dystrophy resulting in markedly impaired vision or blindness at birth. LCA is characterized by an extinguished electroretinogram in infancy, which is thought to be indicative of an early and severe impairment of both the rod and cone photoreceptors in the human retina. Recently, the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene was identified as the fourth causative gene of LCA. AIPL1 encodes a 384 amino acid protein of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within the primate AIPL1 protein, which detects a protein of approximately 43 kDa in human retinal extracts. A screen of human tissues and immortalized cell lines with this antibody reveals AIPL1 to be specific to human retina and cell lines of retinal origin (Y79 retinoblastoma cells). Within the retina, AIPL1 was detected only in the rod photoreceptor cells of the peripheral and central human retina. The AIPL1 staining pattern extended within the rod photoreceptor cells from the inner segments, through the rod nuclei to the rod photoreceptor synaptic spherules in the outer plexiform layer. AIPL1 was not detected in the cone photoreceptors of peripheral or central human retina. This study is the first to suggest that AIPL1 performs a function essential to the maintenance of rod photoreceptor function.

Retinitis pigmentosa (RP) and allied diseases are heterogeneous clinically and genetically. Here we summarize the retinal cell types involved in these diseases, the large number of genes that cause them, and the variety of inheritance patterns that the affected families display. Special consideration is given to unusual inheritance patterns. The aggregate carrier frequency for recessive RP alleles may be as high as 10%.

PURPOSE The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. METHODS The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination. RESULTS Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice. CONCLUSIONS The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.

We designed experiments to evaluate the therapeutic potential of glial cell line derived neurotrophic factor (GDNF) to rescue photoreceptors from genetically determined cell death. Gene transfer of the neurotrophic factor to the retina was achieved via a recombinant adeno-associated virus (rAAV) vector containing the chicken β-actin promoter/immediate early cytomegalovirus enhancer (CBA) driving the human GDNF gene. We delivered AAV-CBA-GDNF to the retinas of an animal model of retinitis pigmentosa, the TgN S334ter-4 rhodopsin line of transgenic rats. Immunohistochemical studies localized AAV-CBA-GDNF-derived recombinant protein to cell bodies, inner segments, and outer segments of photoreceptor cells as well as to retinal pigment epithelial cells. We assessed the effect of viral delivery by morphometric and electroretinographic analysis. These experiments showed that GDNF vector treatment leads to increased rod photoreceptor survival as indicated by morphometric analysis of outer nuclear layer thickness. AAV-CBA-GDNF-treated retinas also demonstrated functional improvement by the substantially increased amplitude of electroretinograms. AAV-CBA-GDNF delivery had a significant rescue effect on photoreceptor degeneration in this animal model.

Vitamin A (all-trans-retinol) plays a fundamental role in the visual cycle. There have been several studies establishing a link between retinal diseases and abnormalities in the absorption, transport and delivery of vitamin A1, 2, 3, 4 and oral vitamin A has been reported to retard the progression of retinitis pigmentosa (RP)1. Large doses of vitamin A administrated orally were used in treatment of Sorsby’s fundus dystrophy (SFD)5 and of a mouse model of Leber congenital6. The studies were based on a hypothesis that the night blindness in these conditions is due to a chronic deprivation of vitamin A at the level of the photoreceptors caused by a thickened membrane barrier between the photoreceptor layer and its blood supply. It was found that vitamin A supplementation could lead to dramatic restoration of photoreceptor function, demonstrating that pharmacological intervention has the potential to improve vision in otherwise incurable genetic retinal degenerations.

PURPOSE: To compare histologic findings in an autopsy eye of an 84-year-old man with advanced retinitis pigmentosa and rhodopsin, Glu181Lys, with two cases of autosomal dominant retinitis pigmentosa (one with rhodopsin, Pro23His, and one with rhodopsin, Cys110Arg) and with a normal control, all of comparable age. METHODS: All eyes were prepared for light and electron microscopy within 6 hours after death. RESULTS: Extensive photoreceptor degeneration was revealed in the eyes with retinitis pigmentosa. Some macular cones showed membranous swirls only in the eye with rhodopsin, Glu181Lys. CONCLUSION: The retinal degeneration caused by rhodopsin, Glu181Lys, can feature membranous swirls in the inner segments of cones in the macula. These swirls have not been reported in other cases of dominant retinitis pigmentosa studied so far, and their pathogenesis remains to be defined.

Theretinal degeneration sloworrdsgene encodes rds/peripherin, an integral membrane glycoprotein in the outer segments of rod and cone photoreceptors. Mice homozygous for a null mutation inrdsfail to develop outer segments and undergo subsequent degeneration of photoreceptors by the apoptotic pathway. Mutations in the humanRDSgene are responsible for several forms of inherited blindness including autosomal-dominant retinitis pigmentosa and macular degeneration. Here, we examined the effects of ectopic Bcl-2 expression in transgenic photoreceptors on the rate of retinal degeneration inrdsmutant mice. We observed an approximately twofold preservation of photoreceptors compared with nontransgenicrdsmutant mice at 3 months. Immunoblot analysis showed similar levels of Bcl-2 in 2-, 3-, and 4-week-old transgenic mice. Expression of Bcl-2 in therdsmouse did not lead to outer segment formation and did not induce cell death. These results suggest that Bcl-2 expression may be an effective therapeutic strategy in humans with mutations inRDSor other genes that affect the integrity of photoreceptor outer segments.

Purpose To study the anamnestic immune response to retinal specific antigens of two patients suffering from a rare triad of retinitis pigmentosa, Coats disease and uveitis. Patients 17-year-old girl presented with an acute episode of panuveitis, and her 19-year-old brother suffered from chronic uveitis. On examination, both patients showed retinal vascular changes and subretinal exudations typical of Coats disease, with bone-spicule pigmentary changes as observed in retinitis pigmentosa. Results All routine examinations were unrevealing. However, the peripheral lymphocytes from these two siblings gave a specific anamnestic response to retinal antigens in vitro. A stimulation index of 4.6 was obtained when the sister's lymphocytes were stimulated with interphotoreceptor binding protein, IRBP--during the acute stage of the uveitis. The brother's lymphocytes showed a stimulation index of 2.7 towards S-Ag during the chronic phase of his uveitic condition. Conclusions These results indicate that autoimmunity towards retinal antigens may play some role in specific types of retinitis pigmentosa. Whether these autoimmune reactions are a primary pathological mechanism or are secondary to the extensive destruction of the photoreceptor layer resulting from the retinitis pigmentosa remains debatable.

Retinitis pigmentosa is an inherited degenerative disease of photoreceptors leading to blindness. A well-characterized model for this disease is provided by the retinal degeneration mouse, in which the gene for the rod cGMP phosphodiesterase is mutated, as in some affected human families. We report that D-cis-diltiazem, a calcium-channel blocker that also acts at light-sensitive cGMP-gated channels, rescued photoreceptors and preserved visual function in the retinal degeneration mouse. The long record of diltiazem prescription in cardiology should facilitate the design of clinical trials for some forms of retinitis pigmentosa.

Although retinal neurons usually last the entire lifetime of an individual, many innate genetic and developmental errors and external stimuli can reduce their longevity leading to loss of visual acuity or blindness. Similarly, the lens, largely composed of denucleated fiber cells must remain transparent for life if vision is to remain clear. Apoptosis of retinal neurons and newly generated lens fiber cells contributes to retinal degeneration and cataract formation, respectively, in both humans and experimental mammals. The apoptosis is triggered by many stimuli in addition to inherited mutations and may be amenable to pharmacologic amelioration. These studies not only provide new clinical insights but also the opportunity to investigate the molecular pathways leading to apoptosis in an organ that is not required for survival. The eye, becomes, therefore, an important organ for evaluation of theories of apoptosis in vivo.

The objective of retinal transplantation is to substitute destroyed or degenerated retina to improve visual function. Photoreceptors and retinal pigment epithelium cells of embryonic and nonembryonic origin have been transplanted into the subretinal space in different animal models. Recently, retinal cells have also been used for transplantation in untreated or immunosuppressed patients with retinitis pigmentosa and age-related macular degeneration. Transplants performed in animals such as the RCS rat have maintained retinal function at the transplantation site for long periods of time, although such transplantations in humans have not proved conclusively, to date, whether there is a real effect or not. One reason for this phenomenon seems to be an immune response to transplanted retinal cells at the transplantation site. The detectable rejection process shows that the posterior part of the eye is not absolutely immunologically privileged and that rejection is a serious problem in human retinal transplantation. Many questions concerning transplantation technique, graft treatment and postoperative treatment will be answered through more intensive experiments and clinical trials regarding the immunology. However, rejection of transplanted material is one of the main reasons why retinal transplantation has not yet proved successful.