Abstract
BackgroundRecessive mutations in guanylate cyclase-1 (Gucy2d) are associated with severe, early onset Leber congenital amaurosis-1(LCA1). Gucy2d encodes guanylate cyclase (GC1) is expressed in photoreceptor outer segment membranes and produces cGMP in these cells. LCA1 patients present in infancy with severely impaired vision and extinguished electroretinogram (ERG) but retain some photoreceptors in both their macular and peripheral retina for years. Like LCA1 patients, loss of cone function in the GC1 knockout (GC1KO) mouse precedes cone degeneration. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells.Methodology/Principal FindingsSerotype 5 AAV vectors containing either a photoreceptor-specific, rhodopsin kinase (hGRK1) or ubiquitous (smCBA) promoter driving expression of wild type murine GC1 were subretinally delivered to one eye of P14 GC1KO mice. Visual function (ERG) was analyzed in treated and untreated eyes until 3 months post injection. AAV-treated, isogenic wild type and uninjected control mice were evaluated for restoration of visual behavior using optomotor testing. At 3 months post injection, all animals were sacrificed, and their treated and untreated retinas assayed for expression of GC1 and localization of cone arrestin. Cone-mediated function was restored to treated eyes of GC1KO mice (ERG amplitudes were ∼45% of normal). Treatment effect was stable for at least 3 months. Robust improvements in cone-mediated visual behavior were also observed, with responses of treated mice being similar or identical to that of wild type mice. AAV-vectored GC1 expression was found in photoreceptors and cone cells were preserved in treated retinas.Conclusions/SignificanceThis is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of GC1 deficiency. Importantly, results were obtained using a well characterized, clinically relevant AAV vector. These results lay the ground work for the development of an AAV-based gene therapy vector for the treatment of LCA1.
Highlights
Leber congenital amaurosis (LCA) is an autosomal recessive group of diseases that represent the earliest and most severe form of all inherited retinal dystrophies
Conclusions/Significance: This is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of Guanylate Cyclase-1 (GC1) deficiency
Immunostaining of GC1 knockout (GC1KO) retinas 3 months posttreatment with AAV5-hGRK1-murine GC1 (mGC1) revealed that this promoter drove robust GC1 expression exclusively in photoreceptor outer segments
Summary
Leber congenital amaurosis (LCA) is an autosomal recessive group of diseases that represent the earliest and most severe form of all inherited retinal dystrophies. Gucy2d encodes the retina- specific protein guanylate cyclase (GC1) which is expressed in both cone and rod photoreceptor disc membranes [2]–[3] and plays a role in the regulation of cGMP and Ca2+ levels within these cells. Levels of cGMP within photoreceptor outer segments rapidly fall due to hydrolysis by cGMP phosphodiesterase (PDE) This reduction of cGMP leads to a closure of cGMPgated channels, reduced Ca2+ influx and hyperpolarization of the cell. This decrease in intracellular Ca2+ stimulates return of lightstimulated photoreceptors to the dark state via its interaction with guanylate cyclase (GC) activating proteins (GCAPs), a family of calcium binding proteins that regulate the activity of GC. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells
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