Matthew J. Annear

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65−/− dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85–180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65−/− dog. This finding has important implications for the treatment of human LCA type II patients.

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Semiconductor diode laser transscleral cyclophotocoagulation for the treatment of glaucoma in horses: a retrospective study of 42 eyes.

OBJECTIVE To evaluate the outcome of diode laser transscleral cyclophotocoagulation (TSCP) for the treatment of glaucoma in horses. PROCEDURE Medical records at The Ohio State University were reviewed. All horses that underwent diode laser TSCP between the years of 1995 and 2007 were included. Preoperative, procedural and clinical follow-up data were collected, and telephone follow-up was performed. RESULTS Forty-two eyes of 36 horses were included. Twenty-four hours prior to surgery mean intraocular pressure (IOP) was 37.17 + or - 13.48 mmHg (42 eyes). Forty-one of 42 eyes (98%) were sighted and 39 of 39 (100%) of eyes were receiving topical glaucoma medication. At 3-5 weeks postoperatively the average IOP was 19.36 + or - 12.04 mmHg (22 eyes). IOP remained significantly lower than pretreatment values at all periods of clinical follow-up (P < 0.05). There was no significant difference in vision outcome, or the requirement for topical glaucoma medication relative to pretreatment values at any follow-up period. Hyphema in 5 of 42 eyes was the only complication noted. Of the 27 eyes seen for clinical follow-up, 2 were enucleated because of refractory elevation of IOP. Mean telephone follow-up was 49 months. Twenty-one of 22 owners contacted (95%) reported that the treatment had been of value, 14 of 22 eyes (64%) were receiving topical glaucoma medication, and 13 of 22 eyes (59%) were considered sighted. CONCLUSIONS Diode laser TSCP aided in the control of IOP and maintenance of vision but did not eliminate the need for topical glaucoma medication during the period of clinical follow-up.

Successful Gene Therapy in Older Rpe65-Deficient Dogs Following Subretinal Injection of an Adeno-Associated Vector Expressing RPE65

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

Gene augmentation trials using the Rpe65-deficient dog: Contributions towards development and refinement of human clinical trials

Dogs with a spontaneous mutation in Rpe65 have been key in the testing of gene augmentation therapy using viral vectors to introduce a normal copy of the Rpe65 gene. These ground-breaking experiments have led to Phase I/II human clinical trials for treatment of Leber congenital amaurosis type II (LCAII).

Early-Onset Progressive Degeneration of the Area Centralis in RPE65-Deficient Dogs.

Purpose Retinal epithelium-specific protein 65 kDa (RPE65)-deficient dogs are a valuable large animal model species that have been used to refine gene augmentation therapy for Leber congenital amaurosis type-2 (LCA2). Previous studies have suggested that retinal degeneration in the dog model is slower than that observed in humans. However, the area centralis of the dog retina is a cone and rod photoreceptor rich region comparable to the human macula, and the effect of RPE65 deficiency specifically on this retinal region, important for high acuity vision, has not previously been reported. Methods Spectral-domain optical coherence tomography, fundus photography, and immunohistochemistry of retinal wholemounts and sagittal frozen sections were used to define the time-course and cell-types affected in degeneration of the area centralis in affected dogs. Results Area centralis photoreceptor degeneration was evident from 6 weeks of age, and progressed to involve the inner retina. Immunohistochemistry showed that RPE65-deficient dogs developed early loss of S-cone outer segments, with slower loss of L/M-cone outer segments and rods. Conclusions Early-onset severe photoreceptor degeneration in the area centralis of dogs with RPE65-deficiency offers a model of the early foveal/perifoveal degeneration in some patients with LCA2. This model could be used to refine interventions aiming to improve function and halt the progression of foveal/perifoveal photoreceptor degeneration.