RESCUE OF THE RD PHENOTYPE USING SOMATIC GENE THERAPY

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DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research program is to understand the biochemical processes in photoreceptor cells that if disrupted, lead to cell dysfunction and degeneration. The model system studied in this program is the retinal degeneration (rd) chicken, the only animal model for inherited retinal disease that possesses a cone-dominant retina. In the previous funding period, the investigators determined that the rd gene encoded photoreceptor guanylate cyclase 1 (GC1), a gene critical for cone and rod phototransduction. The results of their analyses show that the rd chicken is a model for Leber's congenital amaurosis (LCA1), an inherited retinal disease of the retinitis pigmentosa family of retinal degenerations that causes blindness in newborn infants. They propose that the GC1 null mutation in the rd chicken leads to abnormally low levels of cGMP in cone and rod cells, loss of phototransduction, and eventually to photoreceptor degeneration. The goal of the studies outlined in this proposal is to rescue the retinal degeneration phenotype of the rd chicken using somatic gene therapy. They will test the hypothesis that expression of normal GC1 in rd photoreceptor cells is sufficient to restore photoreceptor function and prevent photoreceptor cell death. The aims of this proposal are: (1) to identify fragments of the GCAP1 promoter that are capable of directing expression of reporter genes to photoreceptors in vitro (embryonic chicken retina cell culture) and in vivo (chicken embryos); (2) to examine the ability of selected GCAP1 promoters to drive expression of GC1 in photoreceptor cells in rd/rd retinal cultures; and (3) to rescue the retinal degeneration phenotype in the rd chicken using lentivirus to deliver a GCAP1/GC1 transgene to retinal progenitor or post-mitotic retinal cells. They will determine the transcription start point of GCAP1 and examine the specificity and activity levels of GCAP1 promoter fragments by transient transfection of chicken retinal cell cultures and by viral transduction of stage 9-11 chicken embryos. The ability of the GCAP1 promoter to drive GC1 expression in transiently-transfected rd/rd retinal cultures will be examined by measuring GC1 activity. To assess the effectiveness of the GC1 gene therapy, they will examine (1) the electroretinographic responses of the rd/rd retina, (2) retinal morphology, (3) GC1 expression, and (4) expression profiles of circadian-regulated genes whose normal temporal expression pattern is disrupted in rd/rd retina. Comparisons of rd/rd chickens treated during embryonic development versus at hatching will allow one to determine if the efficacy of the therapy is enhanced if it is administered to the earliest expression of the normal gene in vivo.