Biomedical subjects

J G Flannery

Publications and source records attributed to J G Flannery.

At least 19 recordsLinked to original sources

PubMed2001

Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18.

The goal of these experiments was to evaluate the potential of the fibroblast growth factor family members FGF-5 and FGF-18 to rescue photoreceptors from cell death in retinal degenerative disease. Two strains of transgenic rats, expressing either a P23H or an S334ter rhodopsin mutation, were used as model systems. The neurotrophic growth factors were delivered by subretinal injection of adeno-associated virus vectors, driving expression of the genes with a constitutive CMV promoter. Morphological and functional analyses were performed to determine whether FGF-5 or FGF-18 overexpression could ameliorate cell death in the retina. Immunocytochemistry was used to determine the cellular sites of expression of the factors and to test for up-regulation of FGF receptors due to injection. Significant rescue from photoreceptor cell death was found after injections of vectors expressing either FGF-5 or FGF-18 in the animal models. Increased survival of photoreceptors did not produce a significant increase in electroretinographic responses, however, reflecting either trauma due to the surgery or a suppression of signaling due to expression of proteins. Three weeks after injections, both growth factors were localized to the inner and outer segments of photoreceptors, and the receptors FGFR1 and FGFR2 were also found to be up-regulated in these regions. No visible pathological changes were seen in the FGF-5- or FGF-18-treated eyes. These results indicate that the delivery of either FGF-5 or FGF-18 with adeno-associated virus protects photoreceptors from apoptosis in transgenic rat models of retinitis pigmentosa and that the rescue is probably mediated by conventional receptor tyrosine kinase pathways in photoreceptors.

Animals↗

PubMed2001

Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina.

Viral delivery of neurotrophins or other therapeutic genes is an attractive option for treating retinal degeneration. Regulated expression of these genes in the retina is needed to aid in dose delivery and to promote safety. To evaluate whether tetracycline (tet)-inducible transgenes encapsidated in recombinant adeno-associated viruses (rAAV) can provide controlled gene expression in vitro and in the rat retina, two viruses were constructed: a silencer/activator vector and an inducible doxycycline (dox)-responsive GFP vector. Combinations of these two viruses were subretinally injected into wild-type rats and dox was orally administered through the drinking water. Retinal GFP expression was monitored in vivo with a noninvasive fluorescence imaging method. Eyes were also examined by histology, Western analysis, and electroretinography. Subretinal injection of rAAV efficiently delivers inducible genes to both photoreceptors and retinal pigment epithelial cells. GFP expression was initially observed 1 week postinduction, and GFP protein was undetectable after removal of dox. In uninduced animals, GFP expression was negligible. The dox dosage was varied in vivo and showed a correlation to the level of GFP expression. Thus, transduction of retinal cells with tet-inducible vectors allows for tight regulation of gene expression.

Animals↗

PubMed2001

Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa.

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 beta-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.

Actins↗

PubMed2000

Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

Animals↗

PubMed2000

Ribozyme gene therapy for autosomal dominant retinal disease.

Gene delivery to cells of the retina, particularly to photoreceptor cells, has broad potential both for answering basic questions of retinal biology and for more applied therapeutic purposes. The use of ribozymes as therapy for autosomal dominant retinal diseases is a promising technique, and the theoretical and practical basis for their use is discussed. The process involves designing and testing ribozymes first in vitro and then in animal models of retinal disease. Viral vectors based on the nonpathogenic human adeno-associated virus, when coupled with the strong, rod photoreceptor specific opsin promoter, offer an efficient and nontoxic way to deliver and express ribozymes in photoreceptor cells for long time periods of time. Effective ribozyme-mediated therapy also demands careful in vitro analysis of a ribozyme's ability to efficiently and specifically distinguish between mutant and wild type RNAs. Finally, effective demonstration of therapy in an animal model requires careful analysis of any rescue effect in the retina using multiple criteria, including biochemical, structural and physiological assays. For this purpose, ribozyme therapy in a transgenic rat model of retinitis pigmentosa containing a dominant rod opsin mutation (proline-to-histidine change at position 23) is discussed in detail.

Animals↗

PubMed2000

Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa.

PURPOSE: To determine the extent to which rhodopsin mis-sorting and constitutive activation of the phototransduction cascade contribute to retinal degeneration in a transgenic rat model of retinitis pigmentosa. METHODS: Retinas from transgenic rats expressing truncated rhodopsin (Ser334ter) were examined by light and electron microscopic immunocytochemistry at several time points. Retinal degeneration in transgenic rats raised in darkness was evaluated by quantification of outer nuclear layer thickness and by electroretinography. RESULTS: Mutant rhodopsin was found at inappropriately high levels in the plasma membrane and cytoplasm of Ser334ter rat photoreceptors. When the cell death rate was high this mis-sorting was severe, but mis-sorting attenuated greatly at later stages of degeneration, as the cell death rate decreased. The distributions of two other outer segment proteins (the cGMP-gated channel and peripherin) were examined and found to be sorted normally within the photoreceptors of these rats. Raising Ser334ter transgenic rats in darkness resulted in minimal rescue from retinal degeneration. CONCLUSIONS: Because dark rearing Ser334ter rats results in little rescue, it is concluded that constitutive activation of the phototransduction cascade does not contribute significantly to photoreceptor cell death in this rat model. The nature of the rhodopsin sorting defect and the correlation between the severity of mis-sorting and rate of cell death indicate that truncated rhodopsin may cause apoptosis by interfering with normal cellular machinery in the post-Golgi transport pathway or in the plasma membrane.

Animals↗

PubMed2000

Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2.

PURPOSE: We evaluated adeno-associated virus (AAV)-mediated gene transfer of basic fibroblast growth factor (FGF-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. METHODS: Recombinant adeno-associated virus vector (rAAV) incorporating a constitutive cytomegalovirus (CMV) promoter was used to transfer the bovine FGF-2 gene to photoreceptors. AAV was administered by subretinal injection to transgenic rats (TgN S334ter-4) at postnatal day 15 (P15). Control eyes were uninjected, injected with PBS, or AAV-LacZ. Eyes were examined by histopathology, morphometric analysis, and electroretinography at P60. RESULTS: Expression of recombinant FGF-2 slowed the rate of photoreceptor degeneration. Morphologic studies demonstrated significantly more photoreceptors surviving in eyes injected with AAV-FGF-2 than in controls. Insignificant rescue effects were seen in retinas injected with buffer only. No significant inflammatory response or neovascularization was detected. Electroretinographic (ERG) responses of eyes injected with AAV-FGF-2 were increased compared with uninjected eyes; however, these amplitudes were not significantly larger than eyes receiving an AAV-LacZ control vector. CONCLUSIONS: Transduction of retinal cells with AAV-FGF-2 reduces the rate of photoreceptor degeneration in an S334ter-4 animal model. Despite the lack of significantly increased ERG amplitudes from eyes expressing FGF-2, a greater number of surviving photoreceptors was demonstrated. Delivery of FGF-2 using recombinant AAV has potential as a therapy for retinal degeneration.

Animals↗

PubMed2000

The human cGMP-PDE beta-subunit promoter region directs expression of the gene to mouse photoreceptors.

PURPOSE: We previously demonstrated that 350 bp of the human rod cGMP phosphodiesterase beta-subunit (beta-PDE) gene promoter are sufficient to direct high levels of gene expression in human Y-79 retinoblastoma cells in vitro. In this study the cell specificity and expression pattern conferred by the short beta-PDE 5' flanking sequence in vivo were examined. METHODS: A construct containing the bacterial LacZ gene driven by a fragment of the beta-PDE 5' flanking region (-297 to +53) was used to generate transgenic mice. Gene expression was analyzed by measuring beta-galactosidase activity in tissue homogenates or visualizing enzymatic activity or protein production at a cellular level by in situ histochemistry or immunocytochemistry. RESULTS: Three independently derived transgenic lines were generated carrying the -297 to +53 beta-PDE 5' flanking region fragment. Within the retina, the reporter gene was specifically expressed in photoreceptors, consistent with the localization of endogenous beta-PDE. Significant expression of LacZ was not observed in other ocular or peripheral tissues. CONCLUSIONS: Photoreceptor-specific reporter gene expression is driven in vivo by a 350-bp segment of the beta-PDE 5' flanking sequence. This study demonstrates the utility of the human beta-PDE promoter for directing the expression of foreign genes to photoreceptors and suggests that the -297 to +53 beta-PDE 5' flanking region fragment may have important implications for therapeutic gene delivery to the visual cells.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1998

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

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

Aging↗

PubMed1992

Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Noncontact transscleral neodymium-YAG laser photocoagulation of the pigmented rabbit retina.

We studied the applicability of transscleral delivery of noncontact neodymium-YAG laser in its free-running mode (10 milliseconds) in creating chorioretinal lesions in 16 rabbits. After the laser was applied, the eyes were studied by indirect ophthalmoscopy, light microscopy, and electron microscopy. Lesions generated with low-energy and defocus settings were characterized by intact sclera, variable clumping of the pigmented choroidal lamellae, destruction of choriocapillaris, and loss of the retinal pigment epithelium. In the central region of laser exposure, vacuolization of the retinal pigment epithelium and outer retina were observed. No scleral changes were noted. In contrast with lesions produced with high-energy and/or high-defocus settings, these lesions were produced without hemorrhagic phenomena or breaks in the neurosensory retina. These data demonstrate that noncontact transscleral neodymium-YAG photocoagulation is capable of very localized, selective destruction of the retina and choroid and deserves further study.

Animals↗

PubMed1991

Tissue plasminogen activator treatment of experimental subretinal hemorrhage.

To determine if tissue plasminogen activator, a clot-specific fibrinolytic agent, could eventually be used to assist in the clearance or removal of subretinal hemorrhage, we studied the effect of subretinal injections of tissue plasminogen activator, autologous blood, balanced salt solution, and the combination of either tissue plasminogen activator or balanced salt solution after subretinal injection of autologous blood on retinal morphologic characteristics and clearance of subretinal hemorrhage in the albino rabbit. No morphologic evidence of tissue plasminogen activator toxicity was found in the rabbit retina at a dose of 25 to 50 micrograms/0.1 ml. Subretinal hemorrhage cleared faster after subretinal injection of tissue plasminogen activator when compared to balanced salt solution (P = .0005) but did not completely prevent overlying retinal degeneration. Both tissue plasminogen activator and balanced salt solution were found to decrease the toxic effects of subretinal blood on the morphologic characteristics of the rabbit retina, and this effect can be explained at least partly by dilution of the subretinal blood.

Animals↗

PubMed1991

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Animals↗

PubMed1990

Uptake, processing and release of retinoids by cultured human retinal pigment epithelium.

Upon absorption of a photon, the 11-cis retinaldehyde chromophore of rhodopsin is isomerized and reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments, whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11-cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer: and 11-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase, retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro.

Cells, Cultured↗

PubMed1990

A histopathologic study of a choroideremia carrier.

We have examined eyes from a heterozygote (carrier) of choroideremia, an X-linked disease. Gross examination revealed irregular pigmentation at the level of the retinal pigment epithelium (RPE) except at the posterior pole, and islands of well defined depigmentation of 1-4 mm in diameter in the midperiphery. The optic nerve and retinal blood vessels appeared normal, and there was minimal pigment migration into the retina. Histopathologic examination showed normal photoreceptors in the posterior and anterior fundus, but the outer segments were short or absent in much of the equatorial region. Little gliosis was noted in areas of retinal atrophy. The RPE was abnormal, with irregular thickness and pigmentation associated with variable lipofuscin content from one RPE cell to another, as shown by fluorescence microscopy. There were areas of profound atrophy in the equatorial region, with abrupt transitions between relatively normal RPE and photoreceptors, and retina devoid of RPE and photoreceptors. Bruch's membrane was thickened to a greater extent than is common in age-related change. The choriocapillaris was normal in areas with normal photoreceptors, except for widening of the intercapillary pillars. In those regions with abnormal photoreceptors, choroidal capillaries were fewer in number, had reduced luminal diameter, and fenestrae were sparse. In some areas of intense atrophy, there were no choroidal capillaries. The findings are compatible with the primary defect residing in the RPE. The Lyon hypothesis of X-chromosome inactivation and mosaicism could explain the irregularity of change and areas of intense atrophy, but abrupt demarcation between grossly abnormal, and relatively well preserved retina also occurs in hemizygotes (affected males).

Aged↗

PubMed1989

Degenerative changes in a retina affected with autosomal dominant retinitis pigmentosa.

The eyes of a 17-year-old male donor who was affected with autosomal dominant retinitis pigmentosa with variable expressivity have recently become available for study. Initial macroscopic examination of the fundus revealed bone spicules located in 180 degrees of the postequatorial fundus centered on the inferonasal quadrant. Light microscopic examination of the retina showed degeneration within each quadrant characterized by an absence of rods and cones in the equatorial areas, and the presence of photoreceptors in the more peripheral and central retina. Ultrastructural examination disclosed photoreceptors that were abnormal in all regions when compared to a control eye from a 26-year-old donor. Intact rods were restricted to the peripheral quadrants, and intact cones were identified in the fovea and far periphery. In areas of intermediate degeneration, many outer segments were either shortened and disorganized or absent. Regions of severe degeneration were characterized by the complete loss of the photoreceptors and apposition of the external limiting membrane to the retinal pigment epithelium. The density of rods and cones was found to be substantially lower than normal in all regions. In areas of relatively intact photoreceptor outer segments, we found ultrastructural evidence of recent phagocytic activity, and fluorescence microscopy revealed no unusual accumulation of lipofuscin within the pigment epithelium or subepithelial debris. The choroid and inner retina were normal throughout the eye. The normal condition of the choroid, retinal pigment epithelium, and inner retina implies that the primary disorder resides within the photoreceptor cell.

Adolescent↗