This project is focused on optimizing lentiviral vector mediated gene transfer into long-term repopulating cells, on the evaluation of globin gene vectors, on developing strategies to activate the endogenous globin genes and on evaluating the safety of globin gene therapy approaches. The proposed research involves stem cell targeted gene transfer into autologous cells which will be done in a rhesus transplantation model whereas strategies to optimize globin vectors and to evaluate their safety will be performed using cultured erythroid cells, cell lines and murine models in addition to the rhesus model. We have developed a lentiviral vector system based on simian immunodeficiency virus (SIV) that efficiently transfers genes into cytokine mobilized, peripheral blood stem cells of rhesus macaques yielding approximately 20% genetically modified cells following hematologic reconstitution.
In Specific Aim 1, experiments are proposed with a goal of achieving a high level of transduction efficiency into repopulating stem cells from steady state bone marrow while limiting vector copy number to 1 or as few as possible per transduced cell. Efforts will be also be made to highly enrich stem cells so that the transduced cells infused can be limited to those which have the potential to contribute to long-term repopulation. To evaluate the safety of stem cell-targeted gene transfer, all transplanted animals will be monitored long-term for the behavior of clones of hematopoietic cells containing a vector genome which has integrated near a proto-oncogene.
In Specific Aim 2, experiments are designed to determine whether drug selected hematopoietic stem cells will generate erythroblasts expressing higher levels of vector encoded gamma-globin than non-selected stem cells. Optimized globin vectors and vectors designed to activate the endogenous gamma genes will be evaluated in cultured erythroid cells in an effort to find a vector design which ensures equal to or >20% HbF. Promising therapeutic globin vectors will be tested in vivo in the rhesus model.
In Specific Aim 3, we will test the hypothesis that the vectors containing beta-globin locus control elements will exhibit lineage-restricted activation of surrounding genes in cell lines and primary murine hematopoietic cells that can be blocked or diminished by an insulator. Overall, our research is focused on developing effective approaches for gene therapy of sickle cell disease while simultaneously evaluating the safety of these approaches.
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