Abstract: The present invention provides a method for promoting the survival and/or expansion of hematopoietic stem cells in culture, comprising culturing the cells in a culture including an effective amount of a mpl ligand, (particularly thrombopoietin), a flt3 ligand and interleukin 6 (IL6). Also provided is a method for modifying hematopoietic stem cells comprising contacting a gene delivery vehicle comprising a polynucleotide sequence with a population of hematopoietic stem cells cultured in the presence of an effective amount of thrombopoietin and a flt3 ligand. Additionally methods of restoring hematopoietic capability in a subject are claimed.

Abstract: Transcriptional silencing of transgene expression from Moloney murine leukemia (MoMLV) retroviral vectors has been a hurdle in bringing effective gene therapy to the clinic. The present invention used an optimized transduction protocol for human hematopoietic stem cells (HSC) from mobilized peripheral blood (MPB) to compare MoMLV and mouse stem cell virus (MSCV) vectors, with or without addition of a scaffold attachment region (SAR) from the human interferon-&bgr; gene. To estimate retroviral vector supernatant quality, transgene delivery to CD34+ cells was quantitated 72 hours after transduction using real-time PCR. To estimate the impact of vector backbone and SAR on transgene expression, the percentage of HSC progeny expressing retroviral transgene was compared 72 hours after transduction, and following 5 week stromal culture, or 6-8 week in vivo HSC repopulation assays (SCID-hu bone and NOD/SCID).