The laboratory has finalized a project studying the use of adenoviral gene transfer into retinal pigment epithelial cells (RPE). The object of these studies was to identify a useful tool for short-term, safe gene transfer into the retina for future clinical applications. Human RPE (hRPE) cells in culture have been shown by our group to be readily transduced by adenovirus vectors. However, following transduction, these cells exhibited toxicity related to the viral vectors. Work in the laboratory has demonstrated that hRPE are capable of transcomplementing portions of the adenoviral vectors which are deleted (E1). This transcomplentation results in low level intracellular replication of the vectors. Various toxic proteins of the adenovirus, such as hexon and penton, have been shown to be subsequently expressed and result in a slow cell death of the RPE. Interestingly, this phenomenon of transcomplentation is not seen when adenoviral vectors are placed into RPE cells from other species verifying the result from this laboratory that adenoviral vectors can infect rat RPE, in-vivo, without evidence of cellular toxicity. The speculation is that the human strain of the adenovirus requires transcomplementation from a human protein. This work indicated the importance of screening potential clinical viral vectors on cultured human cells to ensure absence of cellular toxicity and not to rely on animal modeling to detect vector toxicity. Further work has continued on the study of adenoviral vectors containing deletions in other regions of the viral DNA. Those vectors with deletions in the E1/E4 regions, demonstrated absence of any cellular toxicity in human RPE cells. Southern blots revealed absence of viral replication from human RPE cells transduced with these E1/E4-deleted vectors. Additionally, in contrast to E1-deleted adenoviral vectors, E1/E4 vectors exhibited little or no late adenoviral gene expression. Immunohistochemistry on hRPE cells, transduced with the E1/E4 vector, showed no expression of toxic adenoviral proteins such as hexon and penton. This result is also important since these adenoviral proteins are thought to be responsible for the development of an immune response, in humans, against cells exposed to adenoviral vectors. Additional work in-vivo, has demonstrated that E1/E4 vectors can effectively transduce 100% of RPE cells, in the rodent eye, following subretinal injection and that expression can be maintained for six weeks. Expression of the transgene was demonstrated to fall at 8 weeks following injection. PCR analysis of the RPE demonstrated that vector sequences were maintained in the retina over this period of time and Northern blot analysis indicated continued transcription of the transgene in the retina for up to 6 weeks. This work was further extended by experiments conducted on nonhuman primates. Following vitrectomy, posterior hyaloid peel, subretinal injections of 50 ul of adenoviral vector were successfully injected under the retinas of these animals. These results indicated that subretinal delivery of this vector, in an eye anatomically similar to humans, can be attained. None of the treated animals showed any signs of inflammation. All of the above results indicate that the E1/E4 deleted adenoviral vector may be a safe and useful tool for short term gene delivery to the RPE. Possible clinical applications of short term therapeutic protein expression in the retina include treatment of choroidal neovascularization, tumors, regeneration of photoreceptor outer segments to name a few examples. The immediate future of this project will be to study this vector in a model of rodent choroidal neovascularization recently developed in this laboratory.
The aim will be to express angiogenic inhibitors in the retina and measure the ability to retard the growth of new vessel growth. This study has tremendous importance for multiple diseases, most notably age-related macular degeneration, in which choroidal neovascularization can develop and lead to severe visual impairment.
