No abstract

The success of allogeneic transplantation is in part due to the immunotherapeutic effect mediated by the graft. Autologous transplantation is hampered by the absence of this effect, leading to a higher relapse rate. We have conducted a series of studies designed to augment the immunologic activity of the graft after autologous transplant with a view towards introducing an autologous graft-versus-tumor effect that could decrease the rate of relapse after autologous transplant. These studies have included IL-2 activation of marrow followed by post-transplant infusional IL-2, the development of a novel protocol for the generation of highly efficient cytotoxic effector cells, termed cytokine-induced killer (CIK) cells, with broad and potent antitumor activity. In order to determine the potential for generating peptide-specific cytolytic T cells, studies have been conducted upon transducing antigen-presenting cells (APC) with AAV vector-mediated gene transfer, a vector capable of transducing non-proliferating target cells. Transduction of human monocytes and macrophages resulted in high expression of the transduced gene. This latter study forms the basis for determining whether genetic modification of APC can potentiate specific immune responses to tumor-specific gene products. Taken together, these strategies will hopefully increase the therapeutic efficacy of autologous transplantation.

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

No abstract