STROMA-CONDITIONED MEDIUM AND SUFFICIENT PRESTIMULATION IMPROVE FIBRONECTIN FRAGMENT-MEDIATED RETROVIRAL GENE-TRANSFER INTO HUMAN PRIMITIVEMOBILIZED PERIPHERAL-BLOOD STEM-CELLS THROUGH EFFECTS ON THEIR RECOVERY AND TRANSDUCTION EFFICIENCY

Mobilized peripheral blood stem cells (PBSC) are an attractive vehicle for cancer gene therapy. However these stem cells may have a reduced proliferative capacity due to previous cytotoxic chemotherapy treatmen t of the patient. In addition, primitive hematopoietic stem cells (HSC ) from mobilized peripheral blood are almost exclusively quiescent, wh ich makes it hard to induce proliferation in vitro and thus to improve stable transduction of introduced genes into a sufficiently large num ber of primitive stem cells. In this study CD34-selected mobilized PBS C from lymphoma and myeloma patients were used as target cells for ret roviral-mediated gene transfer using a clinically relevant cell-and se rum-free supernatant transduction protocol. We have investigated vario us parameters that may contribute to an improvement of the poor transd uction efficiency of the primitive HSC, including prestimulation time, the use of the carboxy-terminal fibronectin fragment CH-296, as well as stromal cell line conditioned media. Retroviral supernatant transdu ction in combination with CH-296 increased significantly the gene tran sfer efficiency as compared to supernatant alone and made the use of p olycations redundant. Gene transfer of primitive HSC (cobblestone area forming cell (CAFC) week 6) was specifically improved when this proce dure was preceded by a 5-day pre-culture period as compared to a 2-day transduction procedure. However, irrespective of the numerical recove ry, the CAFC week 6 after retroviral transduction produced less long-t erm culture colony-forming cells, suggesting a loss of individual stem cell quality. The addition of stroma-conditioned media during the pre -culture period did not affect the individual CAFC quality or transduc tion efficiency, but increased greatly the recovery of the total numbe r of transduced and untransduced HSC leading to larger grafts containi ng higher numbers of transduced stem cells.