In Vivo Expansion of Transduced Human Erythroid Cells Using an Mpl-Based Cell Growth Switch.

Cell growth switches are engineered signaling molecules that can trigger cell growth in response to artificial ligands such as chemical inducers of dimerization (CIDs). We have previously shown that a cell growth switch comprised of the intracellular portion of the thrombopoietin receptor, Mpl, allows for the CID-dependent, in vivo expansion of genetically modified primary hematopoietic cells in mouse and dog models. Here we report the application of this approach to the in vivo expansion of genetically modified primary human hematopoietic cells using an immune deficient mouse model. A lentivirus vector encoding a CID-activatible deriviative of Mpl (F36VMpl) and a green fluorescent protein (GFP) reporter was used to transduce human cord blood CD34+ cells. Transduced human cord blood CD34+ cells expanded 347–495 fold in cultures containing no added growth factors and 100 nM of AP20187. We proceeded to test CID-responsiveness following transplantation into NOD-SCID-beta 2 microglobulin null mice. Since significant human red cell engraftment persists for only a few weeks post transplantation in this model, mice were evaluated at 3 weeks post transplantation, following a 2 week course of treatment with either CID (AP20187 10 mg/kg/day) or control vehicle alone (without CID). In 2 experiments totalling 42 mice, CID-administration resulted in a significant rise in GFP positive human cells, with the predominant response occuring among human erythroid cells (exp.1: 4.0 fold, P = 0.0003, exp.2: 12.7 fold, P = 0.038). An increase in transduced human erythroid progenitor cells was observed in the spleens (but not the femurs) of CID treated mice. Effects on other hematopoietic lineages were minor and variable. The effect of CID treatment was no longer evident five weeks after the last dose. The restriction of CID-induced cell growth to the erythroid lineage may make this approach well suited for gene therapy applications in sickle cell anemia and beta thalassemia.