Project Description Retinal degeneration is one of the most genetically heterogeneous groups of disorders known, involving over 184 loci. Several ocular gene therapy clinical trials have remarkably demonstrated that gene therapy is a valid approach to treat retinal diseases. Each of these clinical trials and almost every preclinical gene therapy study thus far have utilized viruses as the gene transfer vector. Viruses have significant advantages as gene transfer vectors- primarily their ability to efficiently deliver genes to post-mitotic retinal cells in vivo. However, viruses also have some disadvantages, including induction of host immune responses, a limited transgene capacity, insertional mutagenesis and difficulty in production. Despite these disadvantages, viruses are the current vector of choice in almost all ocular gene therapy studies because of a lack of alternatives. If the above disadvantages could be resolved by the development of non-viral gene transfer vectors that could deliver genes to post-mitotic tissues such as adult retina, it would have substantial impact on the field of preclinical and clinical ocular gene therapy. Unfortunately, non-viral vectors only work efficiently in cell culture or in neonatal retina where mitosis is ongoing. Hence, unlike viruses, non-viral vectors generally fail to rescue animal models of retinal degeneration unless applied in neonatal murine retina - results from which cannot be directly translated to post-mitotic human retina. Recently, we developed a 3.5 Kd peptide (POD) that can form nanoparticles resembling viruses in size (136nm) when complexed with DNA and enable transgene expression in post-mitotic retina. Although gene transfer with POD nanoparticles was not as efficient as with viruses, it was sufficient to enable a short-term delay in retinal degeneration in vivo. This is only one of two studies thus far demonstrating a delay in retinal degeneration in an adult mouse using a non-viral vector. The major limitation of our study was that of short-term transgene expression from POD nanoparticles. The primary objective of this study is to prolong transgene expression from POD nanoparticles by use of nuclear DNA integration or DNA retention elements. The second objective of this study is to improve the efficiency of gene transfer of POD such that it could be more potent and the third objective is to validate the improvements in POD in two relevant animal models of retinal degeneration. The high level of genetic heterogeneity observed in retinal degeneration hampers the timely availability of therapies for patients as each gene and virus combination needs to be developed through a lengthy process. Such approaches are not economically feasible for the >184 loci. Hence, we propose to use POD nanoparticles not to deliver individual genes but instead, genes encoding neurotrophic factors such as to develop a non-viral, non gene-specific approach to treat retinal degeneration. Upon completion of these studies we will have a novel non-viral vector ready for use in clinical trials pending toxicology studies. If successful, these studies would be a paradigm shift in ocular gene therapy.

According to public opinion polls, blindness is the second most feared condition amongst Americans after cancer. Gene therapy for blindness requires the application of viruses that can cause serious adverse events in patients. The objective of this proposal is to develop a non-viral approach to gene therapy. Upon completion, this study will be ready to advance a non-viral gene therapy approach to clinical trials, pending standard toxicology studies as required by the FDA.