Selective engraftment of genetically modified hematopoietic stem cells by a truncated erythropoietin receptor

Genetic modification of hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Transplantation of HSCs, however, requires toxic myeloablation regimens which render this approach questionable for non life-threatening disorders. A potential alternative is the use of transgenes allowing positive selection of HSCs in vivo. We used MLV-derived retroviral vectors and HIV-derived lentiviral vectors to express a truncated form of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. The tEpoR molecule carries a deletion of the 91 carboxy-terminal amino acids, which enhances its proliferative response due to the elimination of a negative regulatory domain. Murine HSCs expressing retrovirally-transferred tEpoR at different levels (1,500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into co-isogenic mouse recipients. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR significantly increase their marrow repopulation capacity upon xenotransplantation into sub-lethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival and differentiation properties. Long-term analysis of serially transplanted mice showed that expression of tEpoR at physiological levels (i.e., comparable with, or slightly higher than, those of the wild-type EpoR in erythroblastic cells) has no effect on steady-state hematopoiesis, and induces no further expansion of transduced cells after the engraftment period. However, significant overexpression of tEpoR (>8-fold the physiological levels) causes mild anemia and erythrocyte morphological abnormalities. These data indicate that expression of tEpoR is a potential alternative for in vivo selection of murine and human repopulating HSCs.