Development of a novel regulatory gene for in vivo & in vitro expansion of transduced hematopoietic stem cellss

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To overcome the low efficiency of gene transfer into hematopoietic stem cells, we have developed a novel strategy for selective expansion of transduced hematopoietic cells. A fusion gene encoding a chimeric receptor (DELTAGCRER) between G-CSF receptor (G-CSFR) and hormone-binding domain (HBD) of estrogen receptor (ER) was constructed as a prototype of "selective amplifier genes (SAG)". G-CSFR was employed as a signal generator and the G-CSF-binding domain was deleted not to respond to G-CSF.ER-HBD was employed as a molecular switch to control the activity of fusion protein in estrogen (E_2)-dependent manner. Although E_2-inducible growth was achieved when BaIF3 cells were transduced with this SAG, it also induced differentiation in transduced 32D cells upon B2 treatment. Since only a growth signal is required, we modified the DELTAGCRER gene to attenuate the differentiation signal. Phe was substituted for Tyr7O3 in the chimeric receptor. When the resultant SAG (DELTAY7O3F-GCRER gene) was expressed in 32D, sustained growth was observed with minimal differentiation upon E_2 treatment. The findings suggest that DELTAY7O3F-GCRBR gene can function as a more desirable SAG.In addition, a mutant ER (TmR), which specifically binds to a synthetic ligand 4-hydroxytamoxifen (Tm), was used as another molecular switch. When GCRTmR was expressed in Ba/F3 cells, Tm-dependent growth was observed with little response to E_2. The GCRTmR/Tm system may be valuable when the SAG method is applied to in vivo amplification of transduced hematopoietic cells, because the influences of endogenous E_2 can be eliminated.