Inherited retinal diseases are a major cause of blindness, and numerous mutations in both retina-specific and nonspecific genes have been identified that cause photoreceptor degeneration. Conventional gene therapy approaches are based on gene addition strategies using viral vectors such as adenovirus (Ad), adeno-associated virus (AAV) and lentivirus. Researchers have demonstrated that virus-mediated gene delivery may be used to successfully delay photoreceptor cell death in animal models of retinal disease; however, the specific endogenous gene mutations are not corrected. Gene targeting allows one to alter genomic sequences at specific chromosomal sites, resulting in permanent gene modifications. Recent reports indicate that AAV can be used to introduce targeted insertions, deletions and substitutions into homologous chromosomal sequences in vitro, with targeting rates that are 4-6 logs higher than those obtained by conventional transfection or electroporation methods. The experiments outlined in this proposal will apply AAV gene-targeting strategies to the retina. The efficacy of virus-mediated repair will be assessed through biochemical, histological and functional assays. Initially studies will be performed in transgenic mutant beta-galactosidase mice to assess if retinal development and viral vector design impact the efficacy of AAV-mediated gene targeting in the retina. Experiments will subsequently be performed in beta-glucuronidase- deficient mice (gusmps/gusmps). These animals will be used to evaluate the therapeutic potential of AAV-mediated gene targeting for RPE disease. The data gathered from these studies may have widespread applications for many inherited pathological conditions and may ultimately lead to the development of therapies that permanently correct fundamental genetic defects responsible for these conditions.
