Efficient transduction and targeted expression of lentiviral vector transgenes in the developing retina

Gene therapy holds great promise as an effective treatment for genetic diseases. Retinal diseases caused by genetic mutations are among the leading causes of blindness and are an excellent place to begin studying the basic principles of gene transfer-based treatments. In addition to understanding the molecular basis of a target disease, perhaps the most difficult steps in the development of somatic gene therapies are engineering a suitable method to deliver therapeutic transgenes to the diseased cells and achieving appropriate levels of expression for extended periods. The primary goal of this study was to develop a lentivirus-based gene delivery vector that can be used to target the expression of a functional, therapeutic transgene to photoreceptor cells in the retina.; Lentiviral vectors derived from the human immunodeficiency virus type I are emerging as the vectors of choice for long-term, stable in vitro and in vivo gene transfer. Several inherited retinal diseases are caused by mutations in genes that are expressed in photoreceptor cells and are required for normal function of these cells. Cell-specific promoters can be incorporated into viral vector gene expression constructs and are used to direct expression of transgenes to specific cell types, and the level of expression of these transgenes is controlled by selecting promoters that possess different intrinsic activity levels.; A self-inactivating lentiviral vector system was used in a novel manner to study the intrinsic activity profiles of promoters that regulate the expression of photoreceptor-specific genes. Using these methods, we were able to identify regions of these promoters that are capable of targeting gene expression to retinal cells during development. Data from these studies provide clues regarding the cis-acting elements that are important for regulating photoreceptor-specific genes in vivo. Furthermore, we have improved the design of and methods of producing lentiviral vectors that will facilitate use of this system for delivering a normal, functional copy of a therapeutic transgene to retinal cells. The results of these studies lay the foundation for future experiments aimed at studying the potential use of lentiviral vector therapies for treating autosomal recessive retinal diseases such as Leber congenital amaurosis type 1 (LCA1).