1. ¹Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
2. ²Department of Laboratory Medicine, Clinical Research Centre, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

Correspondence: Associate Professor MS Dilber, Division of Hematology, Department of Medicine, M54, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm SE-14186, Sweden. E-mail: Sirac.dilber@ki.se

³Current address: Division of Hemato-Oncology, Department of Medicine, Tartu University, Tartu, Estonia and competence centre for cancer research, Tallinn, Estonia.

Received 23 May 2006; Revised 15 August 2006; Accepted 29 August 2006; Published online 3 November 2006.

Abstract

Gene marking can be used to investigate if progenitor cells harvested from patients are contaminated with tumorigenic cells. It can also provide information about the contribution of hematopoietic stem cells to long-term engraftment and about long-term transgene expression from integrated retroviral vectors. In order to study autologous-infused cell contribution to relapse as well as the long-term persistence of the transgene in hematopoietic cells following autologous bone marrow (BM) transplantation for multiple myeloma, we genetically marked autologous CD34⁺ enriched BM or peripheral blood cell grafts of eight myeloma patients using retroviral vectors. Six patients were subsequently transplanted with the marked graft and followed with regular time points of analysis. Briefly, mononuclear cells were harvested by leukapheresis during 2–4 consecutive days following priming with granulocyte–macrophage colony-stimulating factor (GM-CSF) or G-CSF. The CD34⁺ cells separated on Cellpro ceprate avidin-biotin columns were exposed to the G1Na vector coding for neomycin resistance gene at a ratio of five vector particles per cell at three consecutive time points achieving an average transduction efficacy of 2% (0.43–5.1%). The patients were transplanted with a mixture of transduced cells and un-manipulated graft. Vector integration and transgene expression were analyzed by colony assays and polymerase chain reaction. The transgene could be detected for up to 5 years post-transplant in normal BM cells, even in remission following relapse and no side effects related to retroviral gene transfer were observed. There were no marked myeloma cells observed in the patients either in remission or in relapsing disease, which indicates that contribution of infused cells to relapse is unlikely.