AAV-Mediated Gene Therapy Delays Retinal Degeneration in the rd10 Mouse Containing a Recessive PDEß Mutation

Abstract

Purpose:: To test AAV-mediated gene therapy in the rd10 mouse, a natural model of recessive RP due to a PDEß mutation.

Methods:: A PDEß cDNA driven by a small CBA promoter was packaged into rAAV serotype 5 capsids (AAV5-smCBA-PDEß). Pregnant rd10 mice were kept in a dark environment until pups were 14 days old at which time 1 µl of AAV5-smCBA-PDEß was subretinally injected into one eye under dim red light. The animals were then maintained for 2 weeks in darkness before being moved into a normal 12/12 cycling room light environment. The partner eye was injected either with AAV5-smCBA-GFP, or PBS, or remained uninjected. Three weeks following injection, treated rd10 mice were examined by scotopic ERG. Mice were sacrificed 2 days after ERG for biochemical and morphological examination.

Results:: Substantial scotopic ERG signals were recordable only in eyes treated with AAV5-smCBA-PDEß. The average rod-mediated ERG b-wave amplitude in treated eyes was 180 + 20 µV when the stimulus intensity was 2.68cd/cm². Rescue was most evident in eyes that experienced the largest retinal detachment during vector administration (largest retinal coverage of vector) and the least surgical complications. The restored ERGs had typical a-waves and were up to 30% of the b-wave amplitudes seen in control, congenic wild type animals. Strong PDEß expression was observed in photoreceptor outer segments from treated rd10 eyes by immunostaining of retinal sections while PEDß signal was undetectable in untreated rd10 eyes. In addition, western blot analysis confirmed that PDEß protein was expressed in treated eyes. Light microscopic examination showed that the outer nuclear layer of treated rd10 eyes contained up to 10 rows of nuclei. In contrast, there were only 1-3 rows remaining in untreated eyes. Electron microscopic examination showed that up to 50% of the outer segment length was preserved in treated eyes compared with age matched congenic, control eyes. In untreated rd10 eyes there were no outer segments in the central retina and only residual outer segment membrane in the periphery.

Conclusions:: These data demonstrate that P14 administration of AAV5-smCBA-PDEß can prevent retinal degeneration in rd10 mice as reflected by significant structural, biochemical and electrophysiological preservation/restoration. These results serve as a baseline for studying long-term retinal rescue in rd10 mice.

Keywords: gene transfer/gene therapy • photoreceptors • retinal degenerations: hereditary