This pilot project proposes to develop a means by which pluripotent stem cells transduced with the fetal hemoglobin gene can be chemically selected in vivo. We propose that expression in the stem cells of an appropriate human glutathione S-transferase (GST) will confer resistance to the alkylating agent busulfan and allow for stem cell selection. The method proposed takes advantage of the very high susceptibility of stem cells to the alkylating agent busulfan, the importance of glutathione metabolism in terminating the activity of busulfan in vivo, the ability of transfection of cells with glutathione S-transferase to confer resistance to alkylating agents, and the correlation of resistance to hepsulfam (a close chemical relative of busulfan) with GST expression in human breast cancer cell lines. We will identify, through isolation of human GST from liver and placenta and confirmation with E. coli expressed enzyme, the GST most active in busulfan-glutathione conjugation. The investigators in Project I will introduce the corresponding gene together with a LCR gamma globin gene into hematopoietic stem cells, and we will together evaluate the effectiveness of this strategy by assaying resistance of the transduced cells and their ability to form a busulfan glutathione conjugate in comparison to wild type cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL053750-05
Application #
6110424
Study Section
Project Start
1998-09-01
Project End
1999-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Constantinou, Varnavas C; Bouinta, Asimina; Karponi, Garyfalia et al. (2017) Poor stem cell harvest may not always be related to poor mobilization: lessons gained from a mobilization study in patients with ?-thalassemia major. Transfusion 57:1031-1039
Gori, Jennifer L; Butler, Jason M; Kunar, Balvir et al. (2017) Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates. Stem Cells Transl Med 6:864-876
Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia (2016) Optimizing autologous cell grafts to improve stem cell gene therapy. Exp Hematol 44:528-39
Li, Li B; Ma, Chao; Awong, Geneve et al. (2016) Silent IL2RG Gene Editing in Human Pluripotent Stem Cells. Mol Ther 24:582-91
Karponi, Garyfalia; Psatha, Nikoletta; Lederer, Carsten Werner et al. (2015) Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy. Blood 126:616-9
Vierstra, Jeff; Reik, Andreas; Chang, Kai-Hsin et al. (2015) Functional footprinting of regulatory DNA. Nat Methods 12:927-30
Qi, Heyuan; Liu, Mingdong; Emery, David W et al. (2015) Functional validation of a constitutive autonomous silencer element. PLoS One 10:e0124588
Liu, Mingdong; Maurano, Matthew T; Wang, Hao et al. (2015) Genomic discovery of potent chromatin insulators for human gene therapy. Nat Biotechnol 33:198-203
Polak, Paz; Karli?, Rosa; Koren, Amnon et al. (2015) Cell-of-origin chromatin organization shapes the mutational landscape of cancer. Nature 518:360-364
Deyle, David R; Hansen, R Scott; Cornea, Anda M et al. (2014) A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol 21:969-75

Showing the most recent 10 out of 161 publications