Our work has focused on bringing retroviral gene transfer techniques developed in the Hematology Branch into clinical applications targeting human hematopoietic progenitor and stem cells. We also use these techniques to study the biology of abnormal and normal hematopoieis. Over the past year we have continued to enroll patients undergoing autologous stem cell transplantation for aggressive treatment of breast cancer or multiple myeloma into clinical retroviral genetic marking trials. These trials were designed to answer important questions regarding the feasibility of using retroviral vectors to transfer genes into human hematopoeitic stem and progenitor cells, the potential for using bone marrow versus peripheral blood stem and progenitor cells as targets for gene therapy, the pattern and durability of engraftment with bone marrow versus peripheral blood grafts after transplantation, and the source of relapse after aulogous trnasplnatation for these malignancies. 16/20 patients analyzed thus far post-transplant show evidence of hematopoietic cells containing the transferred gene. 5 patients continue to show the presence of the marker gene at one year after transplantation. The level of marked cells is very low, and we are modifiying the techniques used to get vector into the target stem cell to try and increase efficiency. No adverse events related to the gene transfer procedure have been observed. In preclinical in vitro and animal models we are exploring many issues that need to be addressed in order to improve gene transfer efficiency into repopulating stem cells. We are also defining the characteristics of primitive stem cells cultured or """"""""expanded"""""""" ex vivo. We are studying the effect of blocking of inhibitory cytokines such as transforming growth factor beta on our ability to expand and transduce stem cells. We have found that in vivo """"""""priming"""""""" of primate and murine stem cells with the cyotkines G-CSF and stem cell factor improves the transduction of both marrow and blood stem cells. Using retroviral marking in the primate transplantation model, we are studying the effect of ex vivo """"""""expansion"""""""" with cytokines on engraftment after transplantation. We have begun development of vectors with new selectable markers such as green fluorescent protein that may be useful in stem cell targetted gene transfer. We are also exploring the use of bone marrow stromal elements as targets of gene transfer. We have found that these cells can engraft and deliver a biologically-active molecule such as gamma-interferon to hematopoietic precursors. Based on the data we are generating in the clinical marking trials and in our animal studies, we have embarked on two potentially therapeutic protocols: transfer of the multidrug-resistance gene to autologous marrow to confer chemoprotection in breast cancer patients, and transfer of the glucocerebrocidase gene to bone marrow or peripheral blood stem cells to treat Gaucher Disease.
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