GENE TRANSFER IN PHOTORECEPTOR CELLS

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Our long range goal is to establish the molecular basis for gene transfer as a rational approach to prevent or retard the degeneration of photoreceptor cells that is characteristic of hereditary diseases of the outer retina such as Retinitis Pigmentosa (RP). Recent developments suggest that therapeutic intervention may some day be possible, including gene augmentation or replacement aimed at preventing loss of vision. The eventual success of these therapies will depend on the ability of photoreceptor cells to express newly introduced genes. Progress in the field of gene therapy for RP has been slowed by the lack of available vectors which infect mature photoreceptor cells. However, recent developments in the design of a retroviral vector system based on the human immunodeficiency virus (HIV) (termed a lentivirus) have shown stable in vivo gene transfer into terminally differentiated neurons. This proposal represents the collaboration of a molecular biologist and a virologist who will construct an HIV-based vector to carry the gene deficient in the photoreceptor cells of the rd mutant mouse. A retinal cell biologist will evaluate the action of the vector construct both in vitro and in vivo to determine the amount of infectivity and the ability of the transduced gene to function in the photoreceptor cells. This proposal is based on our preliminary results showing improved gene transfer of retrovirus linked to the beta-galactosidase reporter gene into retinal explants from neonatal mice. Construction of a lentiviral vector linked to an opsin promoter and reporter gene will be tested in vitro using dissociated retinal cells and/or retinal explants from neonatal mice. The rd mutant mouse will be used to show that the introduction of a wild-type gene corrects or ameliorates an abnormal phenotype. We will determine whether the deficiency in the beta-subunit of phosphodiesterase (beta-PDE) in PR cells of the rd mouse can be corrected by viral-mediated delivery of the beta-PDE gene. Upon successful completion-of the in vitro studies, injection of the beta- PDE lentiviral construct into the subretinal space of rd will be used to achieve long-term rescue of photoreceptor degeneration in this mutant. Therapeutic effects of beta-PDE will be evaluated by histological examination and by biochemical and molecular biological methods after lentiviral-beta-PDE transduction. When successful, this project may lead to the eventual design of gene therapy protocols to retard photoreceptor degeneration or rescue photoreceptor cells in patients with RP.