The goal of this research is to develop approaches which will allow the proliferative status of genetically modified hematopoietic stem cells to come under pharmacological control. The system involves introducing a gene encoding a fusion protein into hematopoietic cells. The fusion proteins are composed of a cytokine receptor signaling domain linked to one or more copies of the peptide (FKBP12), that permits binding to a drug (FK506) with high affinity. The signaling molecule remains functionally dormant unless forced to engage in dimerization. Dimerization is controlled by adding a dimeric form of FK506, called FK1012, thus mimicking cytokine-activated proliferative signaling. Preliminary studies have used the erythropoietin receptor as a model to show that mitogenic signaling in response to FK1012 can be achieved.
Specific Aim 1 will evaluate whether fusion proteins containing the cytoplasmic domains of c-kit or flt-3 can be activated using FK1012.
Specific Aim 2 will focus on improving the system by a) minimizing the concentration of FK1012 required to achieve proliferative signaling, b) testing other dimerizers, and c) developing modified receptors in which mitogenic signaling domains are present but maturational signaling domains are absent.
Specific Aim 3 will test dimer-inducible proliferative signaling in transgenic mice expressing EpoR/FKBP12 fusion proteins in erythroid cells or c-kit/FKBP12 fusion proteins in stem cells. Stem cells which express c-kit/FKBP12 will be competed against nontransgenic stem cells in the presence of FK1012.
Specific Aim 4 will address potential problems in the development of retroviral vectors and test whether FK1012 can promote the selection of retrovirally transduced hematopoietic cells in vitro and in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052997-02
Application #
2749633
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1997-08-10
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Harkey, Michael A; Kaul, Rajinder; Jacobs, Michael A et al. (2007) Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Stem Cells Dev 16:381-92
Nagasawa, Yasuo; Wood, Brent L; Wang, Linlin et al. (2006) Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal. Stem Cells 24:908-17
Weinreich, Michael A; Lintmaer, Ingrid; Wang, Linlin et al. (2006) Growth factor receptors as regulators of hematopoiesis. Blood 108:3713-21
Blau, C Anthony; Peterson, Kenneth R (2006) Establishment of cell lines that exhibit correct ontogenic stage-specific gene expression profiles from tissues of yeast artificial chromosome transgenic mice using chemically induced growth signals. Methods Mol Biol 349:163-73
Blau, C Anthony; Barbas 3rd, Carlos F; Bomhoff, Anna L et al. (2005) {gamma}-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human {beta}-globin locus yeast artificial chromosome ({beta}-YAC) transgenic mice. J Biol Chem 280:36642-7
Richard, Robert E; Siritanaratkul, Noppadol; Jonlin, Erica et al. (2005) Collection of blood stem cells from patients with sickle cell anemia. Blood Cells Mol Dis 35:384-8
Zhao, Shengming; Weinreich, Michael A; Ihara, Kenji et al. (2004) In vivo selection of genetically modified erythroid cells using a jak2-based cell growth switch. Mol Ther 10:456-68
Richard, Robert E; De Claro, R Angelo; Yan, James et al. (2004) Differences in F36VMpl-based in vivo selection among large animal models. Mol Ther 10:730-40
Richard, Robert E; Weinreich, Michael; Chang, Kai-Hsin et al. (2004) Modulating erythrocyte chimerism in a mouse model of pyruvate kinase deficiency. Blood 103:4432-9
Richard, Robert E; Blau, C Anthony (2003) Small-molecule-directed mpl signaling can complement growth factors to selectively expand genetically modified cord blood cells. Stem Cells 21:71-8

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