Our objective is the successful preclinical development, within five years, of a strategy and procedure that will achieve effective gene therapy for Sickle Cell disease (SCD). To this end, we are melding the activities our research groups, each with a determined research focus on SCD and with a strong specific background in our respective areas of endeavor: one laboratory with expertise in the biophysics and biochemistry of sickling and transgenic animal models of the disease, one laboratory with expertise in the molecular genetics of the globin genes, one laboratory with expertise in the biology of hematopoietic stem cells (HSCs), and two laboratories expert in gene transfer strategies with special emphasis on retrovirus-mediated transduction of HSCs and transfer of globin gene constructs. Our ultimate objective is the complete and sustained reconstitution of the bone marrow of SCD patients with autologous HSCs that have been transduced with a vector whose expressed gene products are effectively anti-sickling in erythroid cells. The immediate aims of the Proposal involve: (1) the development of modified globin gene constructs with efficacious anti- sickling properties, based on our assignment of anti-sickling residues to the gamma- and delta-globin chains, (2) the development of anti-beta/S globin ribozymes, (3) the further development of retroviral vectors carrying the aforementioned anti-sickling gene constructs linked to beta- locus control region (beta-LCR) derivatives, in order to achieve stable and efficient proviral transmission into HSCs and high levels of expression in erythroid cells, (4) the optimization of efficient ex-vivo selection/isolation procedures applicable to our anti-sickling vectors, to achieve long-term bone marrow reconstitution with 100% of transduced HSCs in all transplanted recipients, (5) the development of new retroviral packaging systems capable of infecting non-dividing HSCs at high efficiency, (6) the design and validation of a novel gene transfer strategy, based on retroviral transfer together with site-specific recombination, to make possible the integration of very large DNA fragments into the genome of HSCs at high efficiency, in order to achieve position-independent expression of anti-sickling constructs containing large beta-LCR elements, (7) the use of in vitro methods and transgenic animal models of SCD to validate the effectiveness of our constructs and retroviral vectors, (8) the characterization of the HSCs of SCD patients, (9) the establishment of an in vivo model of SCD involving the engraftment of SCID/NOD mice with bone marrow cells from SCD patients, and the use of this model for validating our anti-sickling approaches, (10) the investigation of the potential for expanding HSCs from SCD patients in bioreactors in vitro, before and after gene transfer, and (11) the development and validation, in the transgenic mouse model of SCD, of strategies for improved gene therapy transplant protocols using sublethal myeloablative regimens.
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