The goal of this proposed research is to explore gene therapy strategies to rescue retinal photoreceptors from degeneration and restore normal visual function in the rd mouse. RP in humans is a collection of hereditary retinal degenerations with an overall incidence of 1 in every 3500 births in the United States. A sub population of patients with autosomal recessive RP (arRP), the most prevalent form, has been linked to mutations of members of the rod phototransduction cascade (cGMP-PDEbeta). Current investigations show that animal models, the rd mouse and the rcd1 Irish setter dog, have RP-like retinal degenerations which have been linked to defects in the cGMP phosphodiesterase beta subunit gene. The C57BL/6J (rd/rd) mouse strain has been shown to have a nonsense mutation in the cGMP PDE beta subunit gene. This mutation, a C to A transversion at codon 347, is theorized to cause a truncation of the normal gene, eliminating more than half of the gene product. The consequence of the truncation in the animal model are consistent with observed phenotypes in humans with RP. Clinically, this disorder produces severe visual loss; abnormal rod and cone electroretinograms, poor dark adaptation thresholds, and intraretinal pigmentation in the mid-periphery signaling the degeneration of retinal tissue. It is our hypothesis that the beta subunit of the rod PDE can be transduced and expressed in the rd mouse photoreceptor and that this expression will reverse the potential for photoreceptor degeneration in the rd mouse model. To test this hypothesis a suitable delivery system for the viral vector will be devised and comparison of adenovirus and retrovirus vectors will be made to assess their relative infectivity of the rod photoreceptors. The cDNA encoding the PDE beta subunit will be subcloned into these vectors and injected into the subretinal space. To assess whether the beta-subunit of PDE is being translated, retinal tissue extracts will be subjected to (1) HPLC and the cGMP hydrolysis activity assayed; (2) SDS-PAGE/Western blot analysis with a polyclonal antibody specific for the rod outer segment cGMP PDE. Photoreceptor integrity will be examined microscopically at post-injection day 7, day 14, and day 21. The eyes will be enucleated, fixed and histologically stained then sectioned. A comparison of normal and non-treated rd affected retinas from aged matched controls will be made to assess the presence of morphological change. Immunocytochemistry will be used to determine where and how many photoreceptors contain the functional PDE. The experiments will demonstrate that PDE can be expressed in the photoreceptors of the rd mouse and will show the localization of the functional PDE complex versus the non-functional alpha and gamma subunits in these animals. Electroretinography (ERG) will be performed on the mice to appraise the efficacy of rescue. It is our contention that the experience gained in the experiments detailed in this proposal will form the basis for future human clinical trials that will attempt to prevent blindness in patients afflicted with RP.
