Principal Investigator/Program Director (Last, first, middle): Kohn, Donald B. Minimal lentiviral vectors for gone therapy of p-thalassemia. Beta-thalassemias, the most common single gone defect in humans, result from absent or decreased _-globin synthesis but are amenable to gone therapy. Recently, lentiviral vectors have been shown to stably transfer the same 13-globin gone and its regulatory elements that were previously unstable in onco-retroviral vectors. Therefore, lentiviruses have unique mechanisms that allow for stable transmission and high level expression of p-globin. For lentiviral vectors to become clinically relevant, they must be unequivocally safe, since they are derived from HIV. The current vectors that have been reported to stably transmit globin cassettes contain significant amounts of the HIV sequences (~31% of the HIV genome). We have successfully tested the latest generation of self-inactivating (SIN) lentiviral vectors where viral long terminal repeat is deleted upon integration into cells, inactivating viral transcription. This situation ideal for the expression of the highly lineage-restricted 13-globin gone and additionally improves biD-safety of gone delivery. We observe high level erythroid-specific expression of GFP, human ?-globin and ferrochelatase, the latter being sufficient to correct the phenotype in erythrocytic protoporphyria in mice. Additionally, these vectors resist proviral silencing. Nevertheless, SIN lentiviruses still carry significant portions of HIV gag and env sequences. The goal of our study will be to construct a cognate minimal SIN vector that will carry the elements required for stable proviral transmission and high level expression of the human [Y-globin gene. The specific aims will be: Construct a series of vectors carrying different elements from gag and env fragments in addition to the 13-globin cassette and evaluate them in mouse erythroleukemia cells; identify elements required for stability and high-level _globin expression. The optimized vectors will be assessed for their ability to correct the thalassemia phenotype in mice and in human bone marrow cells. These studies will: a) identify key lentiviral elements necessary for high level 13-globin expression; b) produce a safe lentiviral vector containing only minimal viral sequences and; c) increase vector payload and allow insertion of additional 13-globin regulatory and/or insulator elements. The aims comprise a focused plan to express therapeutic levels of human _-globin from an optimized lentiviral vector for safe delivery of the J3-globin gone into stem cells, as a prerequisite for future preclinical studies. PHS 398 Page t01 [unreadable] Principal Investigator/Program Director (Last, first, middle): DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY