Gene therapy of sickle cell disease through enhancement of fetal hemoglobin

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The goals of this project are to develop methods to obtain therapeutic levels of engrafted, gamma-globin vector lentiviral vector-transduced hematopoietic stem cells (HSCs) in patients with sickle cell disease (SCD). Additionally, we w/ill study several approaches to augment accumulation of fetal hemoglobin (HbF) resulting from gamma-globin transgene expression in the context ofthe normal endogenous levels ofthe sickle globin chain. Our specific aims are: 1) to obtain therapeutically relevant levels of hematopoietic stem cells (HSCs) transduced with a lentiviral vector capable of high level, gamma-globin expression in progeny erythroid cells, and 2) to develop multifunctional lentiviral vectors to enhance HbF expression. In the first aim, we will use the MGMT selection system to enable selection of gamma-globin vector-transduced HSCs. Substantial progress was made in this area in the last funding period and we believe, with further improvements, HSC selection in a large animal model will be achieved. Additionally, we will investigate whether a novel HOX fusion protein, NUP98-HOXA10, can be used to increase HSC gene transfer and expansion of gamma-globin vector transduced cells for transplantation. In the second specific aim, experiments utilizing an miRNA approach are proposed to reduce the levels of sickle beta globin so to enhance the accumulation of HbF and augment therapeutic efficacy. We also propose to evaluate two approaches for the ability to permanently re-activate expression of the endogenous gamma-globin genes. The first approach will utilize a designer zinc-finger transcription factor which binds to the -117 site in the gamma-globin promoter. We hypothesize this will lead to activation ofthe gamma-globin gene. The second approach seeks to utilize mlRNA-mediated gene expression knockdown of the newly identified gammaglobin transcriptional repressor BCL1 IA, recently described by Dr. Stuart Orkin. Through these efforts, we seek to obtain HSC engraftment levels of at least 20% with globin-vector modified cells and HbF expression in the red cell progeny of 20% of endogenous sickle hemoglobin (HbS) or higher. If these goals can be met, success in a human gene therapy trial for SCD would seem likely RELEVANCE (See instmctions): Our goals are relevant to public health and the mission of the National Heart, Lung and Blood Institute since the development of effective stem cell targeted gene transfer would provide therapy for many inherited blood diseases. Because sickle cell disease causes severe symptoms, disability and often early death, curative therapies such as gene therapy are urgently needed.