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.
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