The study of ocular gene therapy has extended from preclinical work to the preparation of a gene therapy trial for gyrate atrophy (GA). In addition, work on optimization of nonviral approaches to gene delivery into retinal pigment epithelial cells (RPE) has expanded and the study of viral delivery into RPE cells has included examining third generation adenoviral vectors (E1/E4 deleted) and AAV vectors. The study of the correction of the genetic defect in gyrate atrophy has progressed to the institution of a clinical gene therapy trial for these patients. An extension of the work with an E1-deleted adenoviral vector encoding ornithine aminotransferase (hCMV.Ad5.OAT), the mitochondrial matrix enzyme absent in gyrate atrophy, has provided conclusive evidence that the defect in ornithine flux seen in these patients is due to a defect in the one enzyme. In anticipation of a clinical trial for gyrate atrophy, a retroviral vector, lacking a possible immunogenic neomycin resistance gene, and encoding OAT (GCsamOAT) was produced. Using this vector with gyrate atrophy keratinocytes, OAT overexpression was obtained and correction and increase of ornithine flux was achieved. Immunohistochemistry of transduced gyrate atrophy keratinocytes revealed almost 100% transduction efficiency of the retrovirus. Clonal selection of these retrovirally transduced keratinocytes indicated that OAT overexpression was maintained in the lines. Again, 14C-ornithine disappearance from media overlying GCsamOAT transduced gyrate atrophy keratinocytes, was increased compared with that seen with normal keratinocytes indicated an increased rate of ornithine clearance. The clinical trial will involve the transplantation of a five by five centimeter patch of autologous keratinocytes, transduced ex-vivo with GCsamOAT, onto the thighs of patients with gyrate atrophy. Endpoints of the study will include a change in serum ornithine, determination of duration of expression of dermal OAT and examination of possible immune responses of the patients against OAT. Human RPE cells in culture have been shown by our group to be readily transduced by adenovirus vectors. However, following transduction, these cells exhibited toxicity which appeared to be vector related. As stated previously, Southern blot and dot/blot gels revealed a time dependent replication of viral DNA in the absence of complementing E1 sequences. Therefore, in the past year, vectors with both E1 and E4 deletions have been used, both in-vitro and in-vivo, to transduce human RPE cells. The results show that with further deletions in the E4 region, cellular toxicity is markedly reduced while expression is maintained. In addition, similar results were seen with the use of adeno-associated virus. Further work will delineate the safety of these two vectors for future human ocular clinical trials. Because of the cellular toxicity observed in human RPE cells following adenovirus vector transduction, nonviral approaches to gene transfer into these cells have been explored. This work has identified the cationic polymer, polyethylenimine (PEI) as a useful preparation for this work. Efficient transduction (greater than 50%) has been achieved with human RPE cells in culture and work is on-going to determine the ability to transduce these cells in-vivo. Previous work has demonstrated the ability of E1-deleted adenoviral vectors to introduce genes into the RPE cells of rodents. Extension of this work, using an E1-deleted adenoviral vector that expresses vascular endothelial growth factor (VEGF165), has resulted in a rodent model of choroidal neovascularization and subretinal exudation. Further work to examine this model as a potential tool to study age-related macular degeneration is now underway.
