The success of the gene therapy trial in treatment of severe combined immunodeficiency (SCID) provides evidence of the power and utility of in vivo selection of transduced hematopoietic cell populations. Over the past decade, we and others have demonstrated the effectiveness of a combined gene transfer and pharmacologic approach for in vivo selection of hematopoietic stem cells utilizing chemotherapy-resistance genes. In particular, transfer of mutants of O6 methylguanine DNA methyltransferase (MGMT) in combination with inhibitors of this protein, such as 6-benzylguanine (6-BG), and treatment with alkylating agents have been demonstrated to be highly effective for in vivo selection of hematopoietic cells, specifically hematopoietic stem cells. Gene transfer of MGMT has wide application, not only in stem cell selection, but also in generating chemo-resistant hematopoiesis that may allow dose intensification in cancer therapies. While this approach is effective, little is known neither about the potential damage to the functional stem cell compartment nor the long term effects of residual DNA damage present in long-lived cells after such selection. In addition, although the risk of insertional mutagenesis resulting from integrating retroviruses has received wide-spread recent attention and is an important issue facing application of gene transfer to human diseases, we believe that these risks are related not simply to the integration event, but are likely also dependent upon the specific transgene expressed and other factors involving the manipulated cells, such as proliferative stress and proliferative history. In this interactive, multi-investigator grant application, we propose to examine the effectiveness of in vivo stem cell selection, the effects of such selection on long-term stem cell function, the quantity and quality of residual DNA damage that is present after in vivo selection and finally the pathophysiological relevance of the combination of these factors in addition to vector integration on the incidence of leukemia transformation of hematopoietic cells in vivo. The proposed work includes collaborative efforts of a number of accomplished and productive investigators with expertise in hematopoiesis, virus vector design, detection and characterization of DNA damage and vector integration identification/bioinformatics. The research plan directly models and evaluates the effects of in vivo selection and DNA damage on a critical, sensitive, and therapeutically relevant biological compartment (hematopoietic stem cells). The results from these studies will provide powerful insight into the application of virus vectors to genetic diseases and cancer as well as detailed consequences and benefits of protecting hematopoietic stem cells from DNA damage.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Hematopoiesis Study Section (HP)
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Wright, Daniel G
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Children's Hospital Boston
United States
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Muller, Lars U W; Milsom, Michael D; Harris, Chad E et al. (2012) Overcoming reprogramming resistance of Fanconi anemia cells. Blood 119:5449-57
Müller, Lars U W; Williams, David A (2009) Finding the needle in the hay stack: hematopoietic stem cells in Fanconi anemia. Mutat Res 668:141-9
Müller, Lars U W; Daley, George Q; Williams, David A (2009) Upping the ante: recent advances in direct reprogramming. Mol Ther 17:947-53
Milsom, Michael D; Jerabek-Willemsen, Moran; Harris, Chad E et al. (2008) Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells. Cancer Res 68:6171-80
Muller, Lars U W; Milsom, Michael D; Kim, Mi-Ok et al. (2008) Rapid lentiviral transduction preserves the engraftment potential of Fanca(-/-) hematopoietic stem cells. Mol Ther 16:1154-60
Ball, Claudia R; Pilz, Ingo H; Schmidt, Manfred et al. (2007) Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression. Blood 110:1779-87
Will, Elke; Bailey, Jeff; Schuesler, Todd et al. (2007) Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials. Mol Ther 15:782-91
Milsom, Michael D; Williams, David A (2007) Live and let die: in vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection. DNA Repair (Amst) 6:1210-21