Adeno-Associated Viral Mediated Gene Therapy for RPE65 Gene Defect in a Canine Model - Partial Correction of Morphological Phenotype

Abstract

Abstract: : Purpose:Mutations in the RPE65 gene result in an inability to synthesize the 11-cis retinal required for visual pigment formation in the photoreceptor cells. Consequences of RPE65 gene defects are the accumulation of large numbers of retinoid-containing lipid droplets in the RPE and photoreceptor outer segment degeneration. AAV-mediated gene therapy has been shown to partially restore vision in dogs with an RPE65 gene defect. Studies were undertaken to determine whether this restoration of visual function is accompanied by reversal of lipid droplet accumulation in the RPE and restoration of normal photoreceptor cell morphology. Methods:AAV vectors were constructed with either RPE65 or enhanced green fluorescent protein (EGFP) cDNAs linked to a cytomegalovirus promoter. The vectors were injected subretinally into the eyes of dogs with an RPE65 mutation that results in functional blindness. One eye of 6 dogs was injected with the AAV-RPE65 vector and the contralateral eye in 4 of the dogs was injected with the AAV-EGFP vector. The ERG responses of both eyes were monitored over a several month period after which the eyes were enucleated and examined with either fluorescence microscopy (for EGFP) or immunocytochemistry (for RPE65) and with electron microscopy. Results:Both vectors resulted in transduction primarily of the RPE. A small number of EGFP expressing cells were observed in the neural retinas of the AAV-EGFP treated eyes. In the AAV-RPE65 treated eyes, RPE65 immunolabel was restricted exclusively to the RPE. Transduction by both vectors was restricted to region of the eye near the injection site. In the AAV-RPE65 treated eyes, the RPE lipid droplets completely disappeared from the treated area of the retina, but not from parts of the retina distant from the injection site. No restoration of outer segment morphology was observed in the retina overlying the regions where lipid droplet accumulation was reversed. Conclusion:AAV-mediated gene therapy reverses lipid droplet accumulation in the RPE in RPE65 mutant dogs indicating that normal retinoid metabolism has been restored. Although this was sufficient to partially restore visual function, the therapeutic benefit did not appear to be accompanied by reversal of photoreceptor outer segment degeneration.

Keywords: 419 gene transfer/gene therapy • 562 retinal degenerations: hereditary • 571 retinoids/retinoid binding proteins