Boosting the Effects of Gene Therapy in RPE65 Null Mutation Dogs by Re–Treatment

Abstract

Abstract: : Purpose: There is a successive decrease in ERG parameters after gene transfer in RPE65 null mutation dogs, with amplitudes reduced to pre–operative values approximately 36 months after the initial gene transfer treatment. Therefore a study was performed to evaluate the effects of subretinal re–injection of an RPE65 gene construct. Methods: Three dogs, homozygous for the RPE65 null mutation, were included. Affected animals were initially treated subretinally with 70 – 100 µl of rAAV.RPE65 gene construct (2 X 10¹² particles/ml) in the right eye (OD), and 0 – 100 µl of rAAV.GFP (2 X 10¹⁰ transforming units/ml) in the left eye (OS). The dogs were re–treated using similar amounts of the gene construct in OD at 4, 6 and 36 months, respectively, after the initial surgery. Bilateral full–field ERGs were obtained pre and post–surgically as previously described. Funduscopy and subjective assessment of functional vision were also performed. Two of the retreated dogs were euthanized, retinas fixed in 2% paraformaldehyde and stained with antibodies for retinal markers, such as RPE65 and transducin. Results: Clinically there was an improvement in visual behavior after re–treatment 4–6 weeks after the 2^nd injection. Scotopic and photopic ERG b–wave responses were increased. Immunohistochemistry showed RPE65 positive immunolabeling in extensive areas of the RPE of the re–injected eyes, which were more widespread than previously observed after only one subretinal injection. Localized areas of photoreceptor outer segment disruption were observed at the injection sites, however. Conclusions: Subretinal re–injection of previously treated eyes using a gene construct in affected RPE65 null mutation dogs is feasible and does further improve vision. Immunohistochemistry showed extensive immunolabeling using an RPE65 antibody in the treated eye, indicating widespread transduction of RPE65, apparently enhanced by the re–treatment. The only side effects of the treatments were traumatic lesions in the outer retina localized to the specific injection sites

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • electroretinography: clinical