Retroviral-mediated gene transduction of bone marrow-derived stem cells

Retroviral gene transfer was utilized to provide chemotherapy resistance to hematopoietic cells, and to characterize human mesenchymal stem cells (hMSCs) as targets for gene therapy. Myelosuppression is the dose limiting toxicity of nitrosourea chemotherapy, resulting from low levels of the DNA repair enzyme, O{dollar}sp6{dollar}-alkylguanine DNA alkyltransferase, in bone marrow-derived progenitors. High efficiency retroviral-mediated gene transfer of the human alkyltransferase cDNA, methylguanine methyltransferase (MGMT), using a myeloproliferative sarcoma virus (MPSV)-based vector, vM5MGMT, into four separate sources of hematopoietic cells (a CML line (K562), adult mouse, fetal rat, and human CD34+) resulted in increased alkyltransferase expression and increased BCNU survival in K562 cells and primary mouse and human hematopoietic progenitors. BCNU could be used as a selective agent in vitro for MGMT transduced, CD34+ -derived progenitors, and in vivo for increased proportion of mouse progenitors with evidence of MGMT integration, expression, and increased BCNU survival. Thus, MGMT expression can increase the nitrosourea resistance of hematopoietic cells and progenitors, and BCNU can selectively enrich for MGMT transduced hematopoietic progenitors.; hMSCs are osteoprogenitors, and have the potential to differentiate into other mesenchymal lineages. Transduction with vM5LacZ, that contains the LacZ and neo genes, enabled LacZ expression and selective expansion of infected hMSCs in G418. vM5LacZ transduced, G418 selected hMSCs seeded into ceramic cubes and implanted subcutaneously in SCID mice differentiated into osteogenic cells (osteoblasts and osteocytes) that continued to express {dollar}beta{dollar}-galactosidase for at least 9 weeks. hMSCs retrovirally transduced with the human IL-3 cDNA retained in vivo osteogenic potential in ceramic cube implants, and produced detectable hIL-3 in the systemic circulation for at least 12 weeks. Thus, hMSCs are a unique bone marrow-derived progenitor population that can efficiently express proviral genes in differentiated cells for extended times in vivo.; In conclusion, MPSV can direct long-term gene expression in marrow-derived progenitors. This allows in vitro and in vivo BCNU selection of MGMT transduced hematopoietic progenitors, and hIL-3 expression from hMSCs in vivo.