Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery-intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates.

Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

Retinoschisin gene therapy in photoreceptors, Müller glia or all retinal cells in the Rs1h-/- mouse.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9.

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.

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.

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.

Light-triggered rod disc shedding in Xenopus retina in vitro.

The present study was designed to determine whether light-induced rod outer segment disc shedding occurs when the eye is isolated from the rest of the organism. Whole eye explants of Xenopus laevis were maintained in vitro and either exposed to 5 min of light or kept in darkness. In both the in vitro eyes and in vivo controls exposed to the same conditions, a significant degree of disc shedding occurred within 1 hr of the light stimulus. The disc shedding response was larger in the explanted eyes than in the intact animals. In explants exposed to [3H]leucine for 24 hr, a radioactive band formed at the base of rod outer segments. Thus both light-stimulated disc shedding and disc synthesis occur in the eye kept in vitro. The results of this study are compatible with other recently reported results suggesting that rod disc shedding is initiated within the eye.

Circadian disc shedding in Xenopus retina in vitro.

To further examine the endogenous rhythm of disc shedding and phagocytosis observed in several species, adult Xenopus were entrained to a 12 hr light/12 hr dark cycle and then placed in constant darkness. At various times during a 3-day period of constant darkness, eyes were explanted and placed into culture medium, then processed for light and electron microscopy. A clear rhythmicity of disc shedding was observed, with pronounced peaks at the times light onset occurred in the original entrainment cycle. Modification of the HCO3- ion concentration in the medium was found to raise the amplitude of the peak of endogenous disc shedding. Explants maintained in culture medium containing deuterium oxide (a compound known to perturb circadian oscillators) were found to shed with a longer interval between peaks. The addition of the protein synthesis inhibitor, anisomycin, to this preparation suppressed the shedding rhythm. The action of anisomycin was investigated by autoradiographic examination of the pattern of 3H-leucine uptake and protein synthesis by the explant. The findings suggest the presence of a circadian oscillator for rhythmic disc shedding within the amphibian eye.

A diurnal rhythm in opsin content of Rana pipiens rod inner segments.

Quantitative electron microscope immunocytochemistry, employing an antibody specific to opsin, was used to evaluate the amount and location of opsin in Rana pipiens rod photoreceptors throughout a 24 hr light/dark cycle. We found a distinct diurnal rhythm in the density of anti-opsin labeling of the rough endoplasmic reticulum (RER) and Golgi apparatus in the myoid region of the rod inner segment. Opsin labeling of these organelles was lowest at light onset, increasing thereafter by three- to four-fold, and remained high until 2 hr into the dark phase. A fall in labeling density occurred within the following 4 hr, and remained low for the remainder of the dark phase. Our finding of a diurnal rhythm regulating inner segment opsin transport in Rana pipiens contrasts with published observations on outer segment membrane turnover, since it has been shown that the rates of disc formation and disc shedding are governed by environmental lighting alone in this species. These results imply that there is opsin pooling in the inner segment during the first 14 hr of a 24 hr light/dark cycle; thereafter the loss of inner segment opsin due to mobilization of this protein from the Golgi exceeds the rate of formation of new opsin. There was no evidence of accumulation of opsin-containing vesicles near the cilium or in the ellipsoid just prior to light onset. At light onset, prominent opsin labeling was identified at the proximal portion of the outer segment in regions separate from the disc stack. In two separate experiments, additional groups of frogs were killed around the time of light onset and were examined by conventional transmission electron microscopy. Disordered disc membranes were seen at the base of the outer segment which were not in register with the disc stack. These disordered membranes were observed as early as 2 hr before light onset, and were no longer observed by 1 hr after light onset. We suggest that these disordered membranes reflect a step in the biogenesis of new discs, serving as a pool of membrane that forms during the later part of the dark cycle. It appears that light onset triggers the ordering of neatly registered discs from this new membrane, rather than assembly of new membrane from pooled transport vesicles in the inner segment.

Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium.

The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism.

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.

Distribution patterns of photoreceptors, protein, and cyclic nucleotides in the human retina.

The concentration of cGMP, cAMP, protein and the number of cone and rod photoreceptors have been measured in parallel arrays of punches, 3 mm in diameter, taken from each quadrant of normal human retinas. A separate punch containing the fovea and parafoveal region was also analyzed. Eyes were obtained from four male donors ranging in age from 35 to 67 yr. The retina thins considerably from the center to the periphery, and consequently the protein content forms a gradient in the same direction. Similar gradients were observed for cAMP and cGMP concentrations. In all eyes studied, the foveal-parafoveal region had higher levels of cAMP than cGMP. The data was analyzed with the aid of a computer in order to obtain three-dimensional maps of the patterns of distribution of the different parameters. A strong correlation between the areas of higher cone density, non-photoreceptor neurons, and cAMP, and an equally strong correlation between rod distribution and that of cGMP was observed. These maps will serve as baseline data in studies of pathological conditions such as retinitis pigmentosa.

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.

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.

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).

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.

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.

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.

Dimensions of a single word-association in the analyst's reverie.

The reverie, an apparently random series of events occurring in the analyst's consciousness when his attention is evenly suspended, is examined through the expansion of one of its elements, a single word-association. When this expansion is compared with other data in the analysis, the reverie appears to be both continuous and self similar.

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve.

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.