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We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.

Acute graft-vs.-host disease (GVHD) continues to present a barrier to successful allogeneic marrow transplantation. T cell-depletion may prevent severe GVHD but carries an increased risk of graft rejection and relapse posttransplant. Clinical trials have defined the number of lymphocytes associated with sustained engraftment but low risk of significant GVHD (greater than grade I or II skin only) asor = 10(5)/kg. We examined T cell-depletion resulting from positive selection of CD34+ hematopoietic cells with a biotinylated monoclonal anti-CD34 antibody and an immunoaffinity column. Eleven patients (six myeloma and five breast cancer) underwent both peripheral blood stem cell (PBSC) collection and marrow harvest prior to autologous transplantation. One PBSC collection and one-third of each marrow underwent column separation. PBSCs were enriched for CD34+ cells from an initial mean of 1.5 to 53.3%, while marrow went from an initial mean of 2.8 to 65.4%. PBSC were depleted of CD3+ cells from an initial mean of 9.6 x 10(9) to 8.6 x 10(6). Marrow CD3+ lymphocyte content was reduced from an initial mean of 5.6 x 10(9) to 8 x 10(5). Since the column permits quantification and salvage of depleted T cells, its use should allow re-addition of T cell-aliquots associated with minimal risk for GVHD and rejection. In addition, since PBSCs were as readily depleted as marrow, allogeneic PBSC transplant may be feasible using this method.