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      Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis

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          Abstract

          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.

          Abstract

          In the Rpe65 -/- 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.

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          Most cited references 44

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          Gene therapy restores vision in a canine model of childhood blindness.

          The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.
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            Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

            Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.
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              HIV-1 genome nuclear import is mediated by a central DNA flap.

              HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.
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                Author and article information

                Affiliations
                [1 ]Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, Lausanne, Switzerland
                [2 ]Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America
                [3 ]Department of Ophthalmology, Program in Genetics and Tufts Center for Vision Research, Tufts University School of Medicine, Boston, Massachusetts, United States of America
                [4 ]Unit of Clinical Oculogenetics, Jules Gonin Eye Hospital, Lausanne, Switzerland
                [5 ]Retinal Electrodiagnostics Research Group, Department of Ophthalmology II, Eberhard-Karls University, Tübingen, Germany
                [6 ]Laboratory of Retinal Cell Biology, University Hospital, Zürich, Switzerland
                Moorfields Eye Hospital, United Kingdom
                Author notes
                * To whom correspondence should be addressed. E-mail: yvan.arsenijevic@ 123456ophtal.vd.ch
                Contributors
                Role: Academic Editor
                Journal
                PLoS Med
                pmed
                PLoS Medicine
                Public Library of Science (San Francisco, USA )
                1549-1277
                1549-1676
                October 2006
                10 October 2006
                : 3
                : 10
                1592340
                10.1371/journal.pmed.0030347
                05-PLME-RA-0674R3 plme-03-10-08
                17032058
                (Academic Editor)
                Copyright: © 2006 Bemelmans et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 12
                Categories
                Research Article
                Genetics/Genomics/Gene Therapy
                Neuroscience
                Ophthalmology
                Ophthalmology
                Gene Therapy
                Custom metadata
                Bemelmans AP, Kostic C, Crippa SV, Hauswirth WW, Lem J, et al. (2006) Lentiviral gene transfer of Rpe65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis. PLoS Med 3(10): e347. DOI: 10.1371/journal.pmed.0030347

                Medicine

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