Gene Therapy Preserves Inner Retinal Neurons and Their Connectivity in rd10 Mice, a Model of Recessive Retinitis Pigmentosa With PDEβ Mutations

Abstract

Purpose: : To characterize capsid mutant AAV8 (Y733F)-smCBA-PDEβ-mediated gene therapy in the rd10 mouse, a natural model of recessive RP with a PDEβ mutations.

Methods: : A PDEβ cDNA driven by a small CBA promoter was packaged into AAV serotype 8 capsids containing a point mutation in surface-exposed tyrosine residues, AAV8 (Y733F)-smCBA-PDEβ. One µl of this vector was subretinally injected into one eye of rd10 mice at postnatal day 14 under red light. The partner eye remained uninjected. The animals were maintained for 2 weeks in darkness before being moved into 12hr/12hr cycling light. Three or six months later, treated rd10 mice were examined by ERG, followed by antibody staining of retinal sections and examination by confocal microscopy.

Results: : Substantial scotopic and photopic ERG signals were recordable only in treated eyes. Rescue was most evident in eyes with the largest retinal detachment during vector administration and the least surgical complications. Immunocytochemistry confirmed rod and cone rescue in treated eyes up to 6-month. Survival did not appear to decline in the 3 month to 6 month post-treatment time interval. Surviving rods and cones displayed normal morphologies while only one row of scattered, aberrant cones, survived in untreated eyes. In untreated retinas, the second order neurons remodeled upon photoreceptor degeneration, undergoing progressive dendritic atrophy. In contrast, treated rd10 retinas exhibited excellent retention of bipolar and horizontal cell morphologies. Dendrites of rod bipolar cells were well-elaborated and penetrated the cavity of rod spherules. Horizontal cell processes were observed in close proximity to the synaptic terminal of rods and cones. Lamination of the inner retina was totally preserved and all the synaptic markers produced a normal pattern of staining.

Conclusions: : The mutant AAV8 (Y733F)-smCBA-PDEβ vector is highly efficient in targeting photoreceptors and promoting their survival in the long term. Photoreceptor rescue results in preservation of inner retinal neurons morphology and synaptic connectivity. Thus, both photoreceptor degeneration and inner retinal remodeling in rd10 mice can be prevented by timely gene therapy.

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