This proposal is a concerted effort towards the gene therapy of sickle cell disease by three senior research teams: one at the Albert Einstein College of Medicine in New York, one at the Massachusetts Institute of Technology in Cambridge and one at the Terry Fox Laboratory in Vancouver. We are building on the significant advances we are accomplished during the current grant period: the first long-term cure of an animal model by retroviral transfer into hematopoietic stem cells (HSC), the achievement of long-term expression of human beta-globin by retroviral transfer in virtually all transplanted mice with the highest level of expression reported to date, the development of novel anti-sickling globins and ribozymes, and the generation of novel strategies and assays for efficient gene transfer to human HSC and analysis of beta-globin gene expression. In this first competing renewal, we now aim at addressing the remaining central issues through further innovation. We will undertake the synergistic optimization of """"""""super anti-sickling"""""""" globins and ribozymes in conjunction with their in vivo testing in sickle mouse models and the acquisition of new information about the site-specific regulation of expression of transduced globin genes in erythroid cells. We currently have well characterized transgenic sickle mice of expression of transduced globin genes in erythroid cells. We currently have well characterized transgenic sickle mice that express exclusively human HbS (HbS, HbF) with balanced chain synthesis: nevertheless, position effects on the transgenes need to be overcome, and we expect to solve this issue by means of Recombinase-Mediated Cassette Exchange (RMCE), a powerful new procedure we have developed. A key issue that limits the pancellular, position-independent, high-level expression of globin genes following gene transfer is the phenomenon of gene silencing, and an important part of our effort will focus on finding effective remedies. In parallel, we will devise a new generation of viral vectors in order to achieve highly efficient transfer of these silencing-resistant globin constructs in human HSC. Improvements in yield of transduced, transplantable human stem cells, which do not compromise stem cell homing or integrity, are still essential. In a multi-pronged approach, we will use new vectors, new sources of hematopoietic stem cells and new in-vitro manipulation protocols to achieve this aim even in non- myeloablated recipients. In addition, the potential of two novel biologically-based strategies for achieving the selective amplification of genetically modified stem cells in vivo will be evaluated. The driving goal of this Program remains the successful preclinical development of a strategy and methodology that will yield an effective gene therapy protocol for the treatment of sickle cell anemia patients.
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