Outstanding progress has occurred in the last 5 years in the field of gene transfer to in vivo repopulating hematopoietic stem cells (HSC). Nevertheless, the unique requirements of an effective gene therapy for sickle cell anemia (SCA) demand improved efficiencies and yields of transduction to human HSC, more reliable long-term expression in erythroid progeny and strategies for the selective in vivo amplification of transduced HSC. Nevertheless, the unique requirement of an effective gene therapy for sickle cell anemia (SCA) demand improved efficiencies and yields of transduction to human HSC, more reliable long-term expression in erythroid progeny and strategies for the selective in vivo amplification of transduced HSC. This project will address each of these three challenges through a logical sequence of basic mechanistic investigation, evaluation of promising leads using normal human and murine target HSC and erythroid progenitors and their final preclinical validation in studies of transduced murine, baboon and SCA patients' HSC transplanted into syngeneic and immunodeficient hosts. To improve HSC transduction efficiency and yield, we will exploit and develop new sources of HSC (e.g. CD34- cells and cells from non-hematopoietic tissues), explore methods to recapture the in vivo homing ability of cultured HSC, and will examine the use of alternative cytokine treatment protocols as well as multiple and novel vectors (from Project 3). To investigate the problem of position dependence and variegated expression of transduced genes, we will characterize integration sites that are resistant to silencing in erythroid cells to facilitate future improved vector design (to be further developed in Projects 2 and 3). To enable small numbers of transduced HSC to gain dominance in non-myeloablated hosts, we will investigate the kinetics and control of HSC amplification post-transplant and examine the ability of two biologically-based, but mechanistically distinct, strategies to selectively promote transduced HSC self-renewal in vivo. Preclinical objectives are to cure thalassemic and transgenic sickle cell mice (from Core B) using anti-sickling gene therapy approaches (originally developed in Project 1), to achieve a demonstrable therapeutic effect in the erythroid progeny of transduced HSC from SCA patients, and to develop and test clinically applicable using baboon marrow cells.
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