The laboratory has begun to extend previous work on gene transfer into the retinal pigment epithelial cell (RPE) by further examining safe and potentially clinical useful applications of these technologies. Additionally, the laboratory has studied adenoviral vectors as research tools for modeling of age-related macular degeneration. Lastly, the group has finished all preclinical studies and strategies for initiation of the first gene therapy trial for treatment of the blinding condition of gyrate atrophy. 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 was related to the transcomplementation of the E1 region by these cells and subsequent E1-deleted adenoviral replication. Therefore, in the past year, vectors with both E1 and E4 or E2b deletions have been used, both in-vitro and in-vivo, to transduce RPE cells. The results show that while expression is maintained and in some cases increased following further deletion of the early genes, some persistent toxicity remained with the E1/E2b deleted adenoviral vector. However, those vectors with further deletions in the E1/E4 regions, showed almost total absence of any cellular toxicity in human RPE cells. Southern blots revealed absence of viral replication from human RPE cells transduced with the E1/E4 vector. Additionally, in contrast to E1-deleted adenoviral vectors, E1/E4 vectors exhibited little or no late adenoviral gene expression. As a result, in vitro work with these vectors in human cells showed maintenance of normal morphology compared with control cells as well as up to 6 week expression of a Lac-Z reporter gene. Further use of this vector in rodents has shown that expression can be maintained at almost 100% of transduction of RPE cells at 4 weeks following a subretinal injection of the vector. Expression has been shown to be continued for up to 6 weeks. These results indicate that this vector may have usefulness in human use. Therefore work is now underway to study its safety in nonhuman primates. Previous work has demonstrated the ability of E1-deleted adenoviral vectors to introduce genes into the RPE cells of rodents. Because of this ability, vectors expressing several growth factors, thought to be important in the growth of choroidal neovascularization (CNVM) and subsequent formation of subretinal scarring, have been generated. Initial work with a vector expressing vascular endothelial growth factor (VEGF165) showed that overexpression of this growth factor alone resulted in the formation of subretinal serosanguineous exudation in a dose dependent fashion. However, absence of any proliferative response was observed. Transforming growth factor beta (TGF-b1) is another growth factor which has been found in surgical specimens of removed CNVMs. Because the production and activation of this growth factor requires a complex interaction with other proteins, a constitutive active version of the protein, containing mutations at cysteine 223 and 225 (TGF-b1Cys223,225) in an adenoviral vector was produced. Overexpression of this form of the growth factor alone resulted in the absence of any discernible pathology. However, when VEGF16 and TGF-b1223,225 were overexpressed simultaneously by cotransduction of RPE cells in vivo, proliferation of endothelial cells, RPE and fibroblasts was seen in the subretinal space. Histology of this model appears to be similar to autopsy specimens obtained from human with CNVM associated with age-related macular degeneration. Work is now underway to further examine this model and dissect the time course and mechanisms of cellular proliferation. Further work will examine the ability of this model to be used to screen potentially therapeutic agents for efficacy in the treatment of patients with CNVM associated with age-related macular degeneration. 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. Published work has now laid the groundwork for this first clinical gene therapy trial for the treatment of an ocular disease. A GMP grade retrovirus, GCsamOAT, has now been produced. Previous work with this vector has already established its ability to overexpress ornithine aminotransferase in keratinocytes obtained from patients with gyrate atrophy. Additionally, transduction is sufficiently high enough that these cells are able to clear ornithine at a marked increased rate compared with that seen from normal keratinocytes. Additionally, clonal selection of these retrovirally transduced keratinocytes indicated that OAT overexpression was maintained over multiple passages. 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.
