Development of targeted gene therapy vectors for retinal disease

The heterogeneous nature of mutations causing retinal disease makes the development of broadly applicable gene replacement therapies challenging. Therefore, my primary goal was to develop gene transfer vehicles that could be useful for treating a wide variety of retinal dystrophies, regardless of the specific gene, mutation, or cell-type specific defect. To achieve this goal, I capitalized on the natural ability of some viruses to efficiently transfer genetic material to the retina. I selected the human immunodeficiency virus (HIV) and human adeno-associated virus (AAV) as a starting point due to their ability to infect post-mitotic cells of the CNS, low immunogenicity, and stable expression.; Evolutionary pressures do not exist in the retina to restrict viral infection to a particular neural or glial cell class, therefore re-targeting of these viruses is a fundamental and necessary engineering aim for the treatment of retinal disease in a precise manner.; Achieving precisely targeted transgene expression in the retina is of paramount importance for treating retinal disease. In devising broadly applicable gene therapies, not only must one consider the most appropriate cell type to express a therapeutic protein, but care must also be taken to avoid potential off target effects caused by exogenous protein expression in a cell class directly compromised by disease. Photoreceptor based retinal degenerations are the most common cause of blindness, therefore the strategies that I have pursued are directed at either preserving remaining photoreceptor cells or converting other types of surviving retinal neurons into photosensors. The current state of retinal gene therapy vectors, potential disease targets, and basic retinal anatomy is outlined in Chapter 1.; Chapter 2 outlines my first goal, which was to characterize the spatiotemporal expression profile of HIV based vectors in the retina. I hypothesized that if delivered to a young developing retina, HIV vectors would transduce retinal progenitor stem cells prior to photoreceptor differentiation. When these vectors were delivered at various stages of retinal development, a temporal window for transduction of photoreceptors and retinal pigment epithelium (RPE) was discovered. These experiments effectively laid the foundation for later work by defining the cellular tropism of lentiviral (LV) vectors carrying ubiquitous promoters and non-native envelope surface glycoproteins.; The next study, described in Chapter 3, focused on targeting HIV vectors to the predominant retinal glia, Muller cells. The natural supportive role of these cells could hypothetically be harnessed for the expression of secreted therapeutic proteins in order to delay vision loss caused by photoreceptor degeneration. I hypothesized that stable transduction of Muller cells by HIV vectors could be achieved through the appropriate combination of promoter, surface glycoprotein, and injection approach with HIV based vectors. In addition to achieving efficient transgene expression in Muller cells in healthy and diseased retinas, this work also demonstrated that many promoters previously considered to drive expression ubiquitously, in fact displayed a neural or epithelial preference in the retina.; Chapter 4 outlines our initial efforts in converting normally non-photosensitive neurons into photosensors in a diseased retina. With the development of both excitatory and inhibitory photosensitive ion channels, and their expression in simple in vitro neural systems, the potential for restoring light sensitivity to a severely diseased retina became a reality. I hypothesized that AAV could be used to target a photosensitive inhibitory potassium (K+) ion channel to retinal ganglion cells (RGC) in a diseased retina, and that light sensitivity could be restored in the absence of photoreceptor input. In vivo delivery of a modified K+ channel was accomplished with an AAV vector carrying a neural specific promoter, and RGCs were shown...