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Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Nature medicine

genetics, Animals, Genetically Modified, Dependovirus, Disease Models, Animal, Genes, Dominant, Genetic Therapy, Histidine, Photoreceptor Cells, pathology, Point Mutation, Proline, Promoter Regions, Genetic, RNA, Catalytic, biosynthesis, Animals, metabolism, Rats, Rats, Sprague-Dawley, Retinal Rod Photoreceptor Cells, Retinitis Pigmentosa, therapy, Rhodopsin, Rod Opsins

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      Abstract

      Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

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      Most cited references 12

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      Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor.

      Numerous inherited retinal degenerations exist in animals and humans, in which photoreceptors inexplicably degenerate and disappear. In RCS rats with inherited retinal dystrophy, the mutant gene is expressed in the retinal pigment epithelial (RPE) cell, and leads to the loss of photoreceptor cells. Photoreceptors can be rescued from degeneration if they are juxtaposed to wild-type RPE cells in experimental chimaeras or by the transplantation of RPE cells from normal rats. In both cases, the rescue effect extends beyond the immediate boundaries of the normal RPE cells, suggesting trophic action of a diffusible factor(s) from the normal RPE cells. We considered that the fibroblast growth factors, aFGF and bFGF, might have such a trophic role as they are found in the retina and RPE cells; bFGF acts as a neurotrophic agent after axonal injury in several regions of the central nervous system, and bFGF induces retinal regeneration from developing RPE cells. Here we report that subretinal injection of bFGF results in extensive rescue of photoreceptors in RCS rats for at least two months after the injection, and that intravitreal injection of bFGF results in even more widespread rescue, across almost the entire retina. The findings demonstrate for the first time that bFGF can act as a survival-promoting neurotrophic factor in a hereditary neuronal degeneration of the central nervous system.
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        Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector.

        The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.
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          Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

          We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.
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            9701253

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