An ocular gene therapy section is being developed that emphasizes the study of direct ocular gene transfer using E1A-deficient adenovirus constructs, the transfer of genes ex vivo into isolated retinal pigment epithelial cells prior to their transplantation, and the use of ornithine aminotransferase (OAT) transfected keratinocytes to treat patients with gyrate atrophy. A mutant of transforming growth factor beta-1 (TGF-beta*), which is secreted in an active form, has been placed into an adenovirus construct. Studies involving transduction of retinal pigment epithelial cells have sho that these cells can be efficiently transduced at a low multiplicity of infection and are capable of producing high levels of a biologically active TGF-beta*. Adenovirus-TGF-beta* has also been injected into the anterior chamber of rats. Immunohistochemical analysis of these eyes has suggested the presence of secreted TGF-beta*. Human retinal pigment epithelial cells (RPE) have been transfected with plasmids containing beta-galactosidase (beta-gal). Studies are underway to examine the optimal modes of transfection. Parallel studies are being done with isolated rat RPE cells. Both cell lines are capable of growth on thin synthetic basement membrane polymers. These polymers are transplanted, as a single cell layer, into the subretinal space of rats. OAT has been successfully transfected into OAT-deficient fibroblasts (CHO cells) and into keratinocytes, isolated from patients with gyrate atrophy. Studies are presently underway to examine ways to increase OAT levels and activity in these cells.
