Another strategy for stem cell expansion utilizes chimeric gene products, called selective amplifier genes (SAGs). SAGs are composed of a dimerization and a signaling domain, which become activated by specific molecules called chemical inducers of dimerization (CID). Dimerization activates the SAG signaling domains to induce cell proliferation (see Fig. 2). Several variations of this theme have been evaluated for both in vitro and in vivo expansion in response to CID administration.
Initial studies utilized the FK506-binding domain of the immunophilin FKBP12 linked to the intracellular signaling domain of either the erythropoietin or c-kit receptors (53,54). The feasibility of this strategy was demonstrated using an interleukin (IL)-3-dependent cell line; addition of the CID (FK1012) to the media rescued transduced cells from IL-3 deprivation (53,54). The intracellular signaling domain of the thrombo-poietin receptor was evaluated in subsequent studies (55). Murine bone marrow cells transduced with this construct could be expanded ex vivo only in the presence of FK1012. Although multilineage expansion was demonstrated at early time points, megakaryocytic cells dominated the cultures at later time points. CID-mediated expansion of CD34+ cord blood progenitors was achieved in later experiments using a similar construct (56). Whereas the expanded murine cultures favored megakaryocytic differentiation, erythroid cells dominated the CID-expanded human cell cultures.
SAG-mediated HSC expansion has also been evaluated in vivo. Zhao et al. recently demonstrated that SAGs derived from Jak family members may be the key to amplifying specific hematopoietic lineages (57). Experiments carried out with a SAG construct containing the JH1 domain of murine Jak2 linked to a tandem binding site for the CID, AP20187, were evaluated in a murine transplant model. Administration of the CID resulted in a rapid expansion of transduced erythrocytes. However, the effect was shortlived, and the transduced erythrocyte population declined to pretreatment levels after CID withdrawal. Another SAG, consisting of the erythropoietin (EPO) receptor dimer-ization domain fused to the thrombopoietin receptor signaling domain, was recently evaluated in cynomogus macaques (58). Transduced CD34+ cells were transplanted directly into irrigated femurs and humeri in unconditioned animals. In the absence of
CID administration (in this case EPO), 2-30% of colony-forming units (CFU) and less than 0.1% of peripheral mononucleocytes were transgene positive after 1 yr. Peripheral blood marking levels in animals treated with daily injections of EPO peaked at 8-9% over the same time period, with polyclonal marking detected in multiple lineages. However, as seen in previous studies, marked cell percentages returned to baseline levels shortly after each CID treatment (58).
Selective amplification strategies reported to date have demonstrated promising results. However, the marking levels obtained in animal models remain low, and depend on continuous CID administration. It remains unclear whether the chimeric gene products are only effective at expanding less primitive cell populations, or decline as a result of an immune response. However, rather than returning to baseline levels, an immune response would likely clear even the more primitive SAG-expressing cells upon CID withdrawal. One advantage SAG-mediated stem cell expansion has over drug selection schemes is the limited toxicities associated with selection. No obvious adverse events were detected from transgene expression or CID treatment in the animal models. Nevertheless, it will be essential to determine whether cells transduced with these constructs exhibit normal checkpoint controls in response to DNA damage when faced with such strong proliferative signals. This issue needs to be addressed, especially if attempts will be made to link SAG and drug selection strategies. Additional candidates for SAGmediated HSC expansion should come from transcriptional profiling studies underway.
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