Komáromy, András M
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Publication Application of a New Subretinal Injection Device in the Dog(2006-06-01) Komáromy, András M; Varner, Signe E; de Juan, Eugene; Acland, Gregory M; Aguirre, Gustavo DThe use of a new subretinal injection device (RetinaJect™ Subretinal Cannula, SurModics, Inc., Eden Prairie, MN) to access the subretinal space in the canine model was evaluated. Subretinal injections were performed in 33 mongrel dogs between 2 and 52 months of age (median = 9 months). In 5 normal dogs the injection of 150 μl saline or India ink occurred by using a conventional subretinal injection device (CSID) with a 30-gauge anterior chamber irrigating cannula. The sclera had to be surgically exposed and penetrated before the subretinal injection with the CSID could occur. After removing the CSID, the conjunctiva over the sclerotomy site had to be closed. In a second group of 28 dogs [16 normals, 10 RPE65 mutants, and 2 with progressive rod cone degeneration (prcd)], the 25-gauge needle of the RetinaJect™ was used to penetrate the conjunctiva and the sclera. Once the tip of the needle was close to the retinal surface, a 39-gauge polyimide cannula was extended and brought into apposition with the retina for the subsequent subretinal injection of 150 μl saline, India ink, or adeno-associated virus (AAV). No closure of the conjunctiva was required. The animals were clinically monitored between 1 and 59 weeks after surgery. From this second group 25 eyes were harvested for routine histological analysis either immediately after surgery or after a clinical observation time of between 1 and 40 weeks. Both devices provided equally successful access to the subretinal space. The main advantage of the RetinaJect™ was that no surgical dissection was required; this led to a shorter procedure time and milder postoperative conjunctival swelling. In summary, the use of the RetinaJect™ can be recommended as an alternative to the CSID for subretinal injections in dogs.Publication Targeting Gene Expression to Cones With Human Cone Opsin Promoters in Recombinant AAV(2008-07-01) Komáromy, András M; Aguirre, Gustavo D; Alexander, John J; Cooper, Anne E; Chodo, Vince A; Acland, Gregory M; Hauswirth, William WSpecific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.Publication Operating in the Dark: A Night-Vision System for Surgery in Retinas Susceptible to Light Damage(2008-05-01) Komáromy, András M; Aguirre, Gustavo D; Acland, Gregory MA standard operating microscope was modified with a bandpass infrared filter in the light path and infrared image intensifiers for each of the 2 eyepieces. We evaluated this system for subretinal injections in normal control dogs and those with a mutation in the rhodopsin gene. Rhodopsin-mutant dogs are a model for human autosomal dominant retinitis pigmentosa, and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light but not the infrared light. The modified operating microscope provided an excellent view of the ocular fundus under infrared illumination and allowed us to perform subretinal injections in the retinas of the rhodopsin-mutant dogs without any subsequent light-induced retinal degeneration. The first description of light-induced retinal damage showed that exposure of albino rats to visible light intensities ordinarily encountered in the laboratory led to irreversible retinal damage.1 This finding soon was extended to other species, including rabbits2 and monkeys.3 Studies in animal models of retinal degeneration (eg, Royal College of Surgeons rats, ABCA4-mutant mice, or rhodopsin-mutant mice and dogs) have emphasized the interplay between the gene mutation and environmental light and demonstrated acceleration of the disease process by light.4- 10 The effect of environmental light on disease severity in humans with retinitis pigmentosa has been suggested by case reports,11 although definitive proof is lacking. However, patients with class B1 rhodopsin mutations may be at risk of accelerated vision loss with increased light exposures because they have defects in dark (bleaching) adaptation similar to those found in rhodopsin-mutant dogs that demonstrate a high susceptibility to retinal light damage.8,12,13 Many of the emerging therapies for retinal degeneration require the intraocular placement of a reagent or device with the use of an operating microscope.14,15 The damaging effect of the microscope light on the normal retina, even with the appropriate filtering of UV light, has been described previously,16,17 and there is increased emphasis on reducing the intensity and duration of the exposures. Herein, we describe a modification of an operating microscope with an infrared bandpass filter and a night-vision system to perform surgical interventions in the posterior segment of the eyes of rhodopsin-mutant dogs without the risk of photochemical retinal damage. This modification prevents acceleration of the photoreceptor degeneration that occurs with exposure to modest light levels as used in routine clinical practice.8 If humans with retinitis pigmentosa are shown to have similar light damage susceptibility, this microscope modification could be considered for surgical procedures such as the subretinal application of gene therapy vectors.Publication Canine 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; Jacobson, Samuel G; Roman, Alejandro J; Korczykowski, Marc; Hauswirth, William W; Acland, Gregory MBackground 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.