Aleman, Tomas S
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PublicationCalcium Channel Blocker D-cis-Diltiazem Does Not Slow Retinal Degeneration in the PDE6B Mutant rcd1 Canine Model of Retinitis Pigmentosa(2001-02-25) Aleman, Tomas S; Aguirre, Gustavo D; Pearce-Kelling, Susan E; Laties, Alan M; Aleman, Tomas S; Jacobson, Samuel G; Aguirre, Gustavo D; Nickle, Amanda; Laties, Alan M; Jacobson, Samuel G; Acland, Gregory MPurpose: D-cis-diltiazem, a calcium channel blocker, has been reported to enhance photoreceptor survival in the rd mouse, a model of retinitis pigmentosa (RP) resulting from mutation of the PDE6B gene. We tested the hypothesis that diltiazem treatment would similarly rescue the canine rcd1 model of RP, which is also caused by a null mutation in the PDE6B gene. Methods: D-cis-diltiazem was delivered orally twice daily to rcd1 affected dogs beginning at 4 weeks of age; untreated age-matched rcd1 dogs served as controls. At 14 weeks, electroretinograms (ERG) were performed on all animals; 14 dogs were euthanized at this age, and 2 dogs at 25 weeks of age. Eyes were enucleated, fixed, and processed for routine histological examination. Results: No significant differences were found in ERG or histopathologic parameters between diltiazem-treated and untreated rcd1 dogs. Neither rcd1 group showed a rod b-wave; ERGs evoked by single white flashes (dark- or light-adapted) and flicker were also identical between groups. Similarly, treated and untreated animals did not differ in the degree of preservation of the photoreceptor layer, confirmed in cell counts within the outer nuclear layer. Conclusions: Treatment of rcd1 affected dogs with D-cis-diltiazem did not modify the photoreceptor disease when results were assessed using either ERG or histopathologic criteria. The positive photoreceptor-rescue effect of calcium channel blockers reported in the rd mouse was thus not generalizable to another species with retinal degeneration due to mutation in the PDE6B gene. Caution needs to be exerted in extrapolation to the comparable human forms of RP. PublicationLong-Term Restoration of Rod and Cone Vision by Single Dose rAAV-Mediated Gene Transfer to the Retina in a Canine Model of Childhood Blindness(2005-12-01) Aguirre, Gustavo D; Aleman, Tomas S; Cideciyan, Artur V; Aguirre, Gustavo D; Bennett, Jean; Aleman, Tomas S; Cideciyan, Artur V; Bennicelli, Jeannette; Dejneka, Nadine S; Jacobson, Samuel G; Maguire, Albert M; Palczewski, Krzysztof; Hauswirth, William W; Jacobson, Samuel GThe short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations. PublicationSafety in Nonhuman Primates of Ocular AAV2-RPE65, a Candidate Treatment for Blindness in Leber Congenital Amaurosis(2006-08-01) Jacobson, Samuel G; Aguirre, Gustavo D; Aleman, Tomas S; Jacobson, Samuel G; Aguirre, Gustavo D; Cideciyan, Artur V; Boye, Sanford L; Aleman, Tomas S; Komáromy, András M; Conlon, Thomas J; Zeiss, Caroline J; Roman, Alejandro J; Cideciyan, Artur V; Schwartz, Sharon B; Komáromy, András M; Maguire, Albert M; Doobrajh, Michelle; Cheung, Andy Y; Sumaroka, Alexandar; Pearce-Kelling, Susan E; Kaushal, Shalesh; Maguire, Albert M; Flotte, Terence R; Hauswirth, William WLeber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV- RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2. RPE65 in RPE65 -mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vector-related toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2. RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2. RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina. PublicationCanine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation(2007-06-26) Aguirre, Geoffrey K; Komáromy, András M; Cideciyan, Artur V; Brainard, David H; Aleman, Tomas S; Avants, Brian B; Gee, James C; Aguirre, Geoffrey K; Komáromy, András M; Aguirre, Gustavo D; Cideciyan, Artur V; Jacobson, Samuel G; Brainard, David H; Aleman, Tomas S; Roman, Alejandro J; Avants, Brian B; Gee, James C; Korczykowski, Marc; Hauswirth, William W; Acland, Gregory M; Jacobson, Samuel GBackground RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA). Methods and Findings RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation. Conclusions Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.