A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

“…Selective expansion was not observed in the other two monkeys that received hydroxytamoxifen (D3 and D4), yet the serum concentrations of hydroxytamoxifen did not reach the biologically optimal levels (at least 100 nM). 24 Serious adverse effects were not observed in the animals after drug treatment, but there were some symptoms associated with high-dose administration of estradiol. Monkeys receiving estradiol (S1 and S2) showed genital swelling.…”

“…22 We previously developed a chimeric selective amplifier gene (SAG) composed of the signalling domain of the granulocyte colony-stimulating factor (G-CSF) receptor and the estrogen receptor hormone-binding domain, and demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen or tamoxifen in vitro. 18,23,24 The estrogen receptor-mediated dimerization of the chimeric gene product is assumed to be critical for the activation of the G-CSF receptor signaling. 25 We have further shown that in vivo expansion of transduced cells with the SAG is feasible in a murine transplantation model.…”

“…23 We constructed a further modified SAG in which a point mutation (G525R) was introduced into the estrogen receptor moiety of the GCRER (Figure 1). 24,28 This SAG product (tamoxifen-responsive SAG, or designated GCRTmR) no longer reacts to endogenous estrogen but reacts to synthetic hormones such as tamoxifen (Tm). 24 To facilitate monitoring in vivo, we constructed a bicistronic retroviral vector expressing both GCRER (as the first cistron) and green fluorescent protein (GFP; as the second cistron) (Figure 1).…”

“…29 Retroviral vectors expressing only GCRER, GCRTmR, or GFP were also constructed ( Figure 1). 24 Additionally, we prepared control, non-expressing retroviral vectors (G1PLI and G1PLII) which contain nontranslated sequences from the neomycin resistance and ␤-galactosidase genes by disrupting the first ATG codons. 30 Single marking study To increase the likelihood of primitive hematopoietic cell cycling and thus retroviral transduction, stem cell factor (SCF) and G-CSF were administered to the animals before bone marrow harvesting.…”

“…In the tamoxifen-responsive SAG, a point mutation (G525R) was introduced into the estrogen receptor moiety so that the molecular switch no longer responds to endogenous estrogen, thus minimizing the background signal transduction generated by endogenous estrogen. 24 Until now, no leukemogenesis has been observed even in the monkeys (S1, S2 and D1) which received the prototype estrogen-responsive SAG (GCRER). Thus, leukemogenesis may be regarded as an unlikely event.…”