Beltran, William
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Publication BEST1 Gene Therapy Corrects a Diffuse Retina-Wide Microdetachment Modulated by Light Exposure(2018-03-20) Guziewicz, Karina E; Cideciyan, Artur V; Beltran, William A; Komáromy, András M; Guziewicz, Karina E; Cideciyan, Artur V; Aguirre, Gustavo D; Ruthel, Gordon; Komáromy, András M; Dufour, Valerie L; Swider, Malgorzata; Jacobson, Samuel G; Sumaroka, Alexander; Kendrick, Brian T; Ruthel, Gordon; Chiodo, Vince A; Heon, Elise; Hauswirth, William W; Jacobson, Samuel GMutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies.Publication Age-Dependent Disease Expression Determines Remodeling of the Retinal Mosaic in Carriers of RPGR Exon ORFn15 Mutations(2009-08-01) Beltran, William; Aguirre, Gustavo D; Beltran, William; Aguirre, Gustavo D; Acland, Gregory MPURPOSE. To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS. Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS. A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared so that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (≥8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina. In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age; however, only patchy degeneration was seen by 1.4 years and was still apparent at 7.8 years. CONCLUSIONS. The time of onset and the progression of the disease differed between the two models. In the early-onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late-onset form (XLPRA1), patches of disease persisted until later ages.Publication Décollement Rétinien Associé à une Cataracte Unilatérale [Retinal Detachments Associated with Unilateral Cataracts](2001-06-01) Beltran, William; Beltran, William; Jégou, Jean-PierreLe décollement de rétine rend la chirurgie de la cataracte inutile : il doit donc être recherché lors de l'examen préopératoire. La technique de choix dans le diagnostic de cette affection est l'échographie oculaire. L'étude de huit cas révèle l'intérêt de cet examen dans le diagnostic des décollements de rétine associés à la cataracte chez le chien à travers la variété des images échographiques obtenues.Publication Development and Validation of a Canine-Specific Profiling Array to Examine Expression of Pro-Apoptotic and Pro-Survival Genes in Retinal Degenerative Diseases(2012-01-01) Genini, Sem; Beltran, William; Genini, Sem; Beltran, William; Aguirre, Gustavo DWe developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of the profiling array was performed with RNA extracted from cultured MDCK cells in the presence or absence of treatment with 10 μM staurosporin for 5 or 10 h. The vast majority of the genes showed positive amplifications, and a number of them also had fold change (FC) differences > ±3 between control and staurosporin-treated cells. To conclude, we established a profiling array that will be used to identify differentially expressed genes associated with photoreceptor death or survival in canine models of retinal degenerative diseases with mutations in genes that cause human inherited blindness with comparable phenotypes.Publication Intravitreal Injection of Ciliary Neurotrophic Factor (CNTF) Causes Peripheral Remodeling and Does Not Prevent Photoreceptor Loss in Canine RPGR Mutant Retina(2007-04-01) Beltran, William; Aguirre, Gustavo D; Beltran, William; Aguirre, Gustavo D; Wen, Rong; Acland, Gregory MCiliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 μg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the central and mid-peripheral retina. CNTF also caused ocular side-effects and morphologic alterations in the periphery that were consistent with cell dedifferentiation and proliferation. Our findings suggest that some inherited forms of retinal degeneration may not respond to CNTF's neuroprotective effects.Publication rAAV2/5 Gene-Targeting to Rods: Dose-Dependent Efficiency and Complications Associated With Different Promoters(2010-09-01) Beltran, William; Aguirre, Gustavo D; Beltran, William; Aguirre, Gustavo D; Boye, Sanford L; Boye, Shannon E; Chiodo, Vince A; Lewin, Alfred S; Hauswirth, William WA prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg ml–1 were used.Publication Assessment of Visual Function and Retinal Structure Following Acute Light Exposure in the Light Sensitive T4R Rhodopsin Mutant Dog(2016-05-01) Aguirre, Gustavo D; Iwabe, Simone; Beltran, William A; Ying, Gui-shuang; Beltran, William AThe effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4–5.5 months) were used in this study: 3 homozygous mutant RHOT4R/T4R, 8 heterozygous mutant RHOT4R/+, and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHOT4R/T4R) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm2 , 170 lux; 0.5 mW/cm2 , 820 lux; or 1 mW/cm2 , 1590 lux) for 1min. All 8 heterozygous (RHOT4R/+) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHOT4R/T4R and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2–36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHOT4R/T4R retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHOT4R/T4R retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHOT4R/T4R retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/ tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.Publication Exclusion of the Unfolded Protein Response in Light-Induced Retinal Degeneration in the Canine T4R RHO Model of Autosomal Dominant Retinitis Pigmentosa(2015-02-19) Genini, Sem; Aguirre, Gustavo D; Marsilli, Stefania; Genini, Sem; Aguirre, Gustavo D; Beltran, William; Sudharsan, Raghavi; Gingrich, Jeremy; Beltran, WilliamPurpose To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHOdog). Methods The left eyes of T4R RHOdogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure. Results A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3. Conclusion The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.Publication Successful Arrest of Photoreceptor and Vision Loss Expands the Therapeutic Window of Retinal Gene Therapy to Later Stages of Disease(2015-10-27) Beltran, William A; Cideciyan, Artur V; Beltran, William A; Cideciyan, Artur V; Aguirre, Gustavo D; Iwabe, Simone; Swider, Malgorzata; Kosyk, Mychajlo S; McDaid, Kendra S; Martynyuk, Inna; Ying, Gui-shuang; Shaffer, James; Jacobson, Samuel G; Boye, Sanford L; Lewin, Alfred S; Hauswirth, William W; Jacobson, Samuel GInherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.Publication Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina?(2007-11-01) Beltran, William; Gu, Danian; Beltran, William; Aguirre, Gustavo D; Li, Zexiao; Acland, Gregory MPURPOSE. The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. METHODS. Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. RESULTS. Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. CONCLUSIONS. The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.